RNA interference mediated inhibition of placental growth factor gene expression using short interfering nucleic acid (SINA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating placental growth factor (e.g., PGF-1 or PlGF-1, PGF-2 or PlGF-2, and/or PGF-3 or PlGF-3) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of placental growth factor gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of placental growth factor genes.

This application is a continuation of U.S. patent application Ser. No.10/922,761, (now abandoned), filed Aug. 20, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/683,990,(now abandoned), filed Oct. 10, 2003, which is a continuation-in-part ofInternational Patent Application No. PCT/US03/05022, filed Feb. 20,2003, which claims the benefit of U.S. Provisional Application No.60/393,796, filed Jul. 3, 2002 and U.S. Provisional Application No.60/399,348 filed Jul. 29, 2002, and parent U.S. patent application Ser.No. 10/922,761 is also a continuation-in-part of International PatentApplication No. PCT/US04/16390, filed May 24, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/826,966,(now abandoned), filed Apr. 16, 2004, which is continuation-in-part ofU.S. patent application Ser. No. 10/757,803, filed Jan. 14, 2004, whichis a continuation-in-part of U.S. patent application Ser. No.10/720,448, filed Nov. 24, 2003, which is a continuation-in-part of U.S.patent application Ser. No. 10/693,059, (now abandoned), filed Oct. 23,2003, which is a continuation-in-part of U.S. patent application Ser.No. 10/444,853, filed May 23, 2003, which is a continuation-in-part ofInternational Patent Application No. PCT/US03/05346, filed Feb. 20,2003, and a continuation-in-part of International Patent Application No.PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit ofU.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002, U.S.Provisional Application No. 60/363,124 filed Mar. 11, 2002, U.S.Provisional Application No. 60/386,782 filed Jun. 6, 2002, U.S.Provisional Application No. 60/406,784 filed Aug. 29, 2002, U.S.Provisional Application No. 60/408,378 filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293 filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129 filed Jan. 15, 2003. The instantapplication claims the benefit of all the listed applications, which arehereby incorporated by reference herein in their entireties, includingthe drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing51USCNT”,created on Jul. 10, 2008, which is 133,088 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of placental growth factor(for example, PGF-1, PGF-2, and/or PGF-3 and corresponding receptors)gene expression and/or activity. The present invention is also directedto compounds, compositions, and methods relating to traits, diseases andconditions that respond to the modulation of expression and/or activityof genes involved in PGF-1, PGF-2, and/or PGF-3 gene expression pathwaysincluding PGF receptors or other cellular processes that mediate themaintenance or development of such traits, diseases and conditions.Specifically, the invention relates to small nucleic acid molecules,such as short interfering nucleic acid (siNA), short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and shorthairpin RNA (shRNA) molecules capable of mediating RNA interference(RNAi) against PGF-1, PGF-2, and/or PGF-3, such as PGF-1, PGF-2, and/orPGF-3 gene expression. Such small nucleic acid molecules are useful, forexample, in providing compositions for treatment of traits, diseases andconditions that can respond to modulation of PGF-1, PGF-2, and/or PGF-3expression in a subject, such as cancer, proliferative diseases,disorders, or conditions, ocular diseases, disorders, or conditions,dermatological diseases, disorders, or conditions, rheumatoid arthritis,endometriosis, and kidney disease.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double-strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy(2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of an siRNAduplex is required for siRNA activity and that ATP is utilized tomaintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certainsingle-stranded siRNA constructs, including certain 5′-phosphorylatedsingle-stranded siRNAs that mediate RNA interference in HeLa cells.Harborth et al., 2003, Antisense & Nucleic Acid Drug Development, 13,83-105, describe certain chemically and structurally modified siRNAmolecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certainchemically and structurally modified siRNA molecules. Woolf et al.,International PCT Publication Nos. WO 03/064626 and WO 03/064625describe certain chemically modified dsRNA constructs. Koninckx et al.,International PCT Publication No. WO 03/63904, describe certaininhibitors of hypoxia induced genes, including certain siRNAs targetingPlGF, to prevent post-operative adhesion formation.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating placental growth factor, such as PGF-1, PGF-2, and/orPGF-3, gene expression using short interfering nucleic acid (siNA)molecules. This invention also relates to compounds, compositions, andmethods useful for modulating the expression and activity of other genesinvolved in pathways of placental growth factor gene expression and/oractivity, such as receptors of PGF-1, PGF-2, and/or PGF-3, by RNAinterference (RNAi) using small nucleic acid molecules. In particular,the instant invention features small nucleic acid molecules, such asshort interfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules and methods used to modulate the expression ofplacental growth factor (e.g., PGF-1, PGF-2, PGF-3, and/or correspondingreceptors) genes.

An siNA of the invention can be unmodified or chemically-modified. AnsiNA of the instant invention can be chemically synthesized, expressedfrom a vector or enzymatically synthesized. The instant invention alsofeatures various chemically-modified synthetic short interfering nucleicacid (siNA) molecules capable of modulating placental growth factor(e.g., PGF-1, PGF-2, PGF-3, and/or corresponding receptors) geneexpression or activity in cells by RNA interference (RNAi). The use ofchemically-modified siNA improves various properties of native siNAmolecules through increased resistance to nuclease degradation in vivoand/or through improved cellular uptake. Further, contrary to earlierpublished studies, siNA having multiple chemical modifications retainsits RNAi activity. The siNA molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic, veterinary,diagnostic, target validation, genomic discovery, genetic engineering,and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofplacental growth factor (e.g., PGF-1, PGF-2, PGF-3, and/or correspondingreceptors) genes encoding proteins, such as proteins comprisingplacental growth factor (e.g., PGF-1, PGF-2, PGF-3, and/or correspondingreceptors) associated with the maintenance and/or development of cancer,proliferative diseases, disorders, or conditions, ocular diseases,disorders, or conditions, dermatological diseases, disorders, orconditions, rheumatoid arthritis, endometriosis, and kidney disease,such as genes encoding sequences comprising those sequences referred toby GenBank Accession Nos. shown in Table I, referred to herein generallyas placental growth factor-1 or PGF-1. The description below of thevarious aspects and embodiments of the invention is provided withreference to exemplary placental growth factor-1 gene referred to hereinas PGF-1. However, the various aspects and embodiments are also directedto other PGF-1 genes, such as homolog genes and transcript variants, andpolymorphisms (e.g., single nucleotide polymorphism, (SNPs)) (e.g.,PGF-2 and PGF-3) associated with certain PGF-1 genes, and genes encodingreceptors of PGF-1, PGF-2, and/or PGF-3. As such, the various aspectsand embodiments are also directed to other genes that are involved inPGF-1 mediated pathways of signal transduction or gene expression thatare involved, for example, in the maintenance or development ofdiseases, traits, or conditions described herein. These additional genescan be analyzed for target sites using the methods described for PGF-1genes herein. Thus, the modulation of other genes and the effects ofsuch modulation of the other genes can be performed, determined, andmeasured as described herein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a placental growth factor (e.g., PGF-1) gene, wherein said siNAmolecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of aPGF-1 RNA via RNA interference (RNAi), wherein the double-stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 28 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the PGF-1 RNA for the siNA molecule to directcleavage of the PGF-1 RNA via RNA interference, and the second strand ofsaid siNA molecule comprises nucleotide sequence that is complementaryto the first strand.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of aPGF-1 RNA via RNA interference (RNAi), wherein the double-stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 23 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the PGF-1 RNA for the siNA molecule to directcleavage of the PGF-1 RNA via RNA interference, and the second strand ofsaid siNA molecule comprises nucleotide sequence that is complementaryto the first strand.

In one embodiment, the invention features a chemically synthesizeddouble-stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a PGF-1 RNA via RNA interference (RNAi), whereineach strand of the siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the PGF-1 RNA for the siNAmolecule to direct cleavage of the PGF-1 RNA via RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble-stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a PGF-1 RNA via RNA interference (RNAi), whereineach strand of the siNA molecule is about 18 to about 23 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the PGF-1 RNA for the siNAmolecule to direct cleavage of the PGF-1 RNA via RNA interference.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a PGF-1 gene, for example, wherein thePGF-1 gene comprises PGF-1 encoding sequence. In one embodiment, theinvention features an siNA molecule that down-regulates expression of aPGF-1 gene, for example, wherein the PGF-1 gene comprises PGF-1non-coding sequence or regulatory elements involved in PGF-1 geneexpression.

In one embodiment, an siNA of the invention is used to inhibit theexpression of PGF-1 genes or a PGF-1 gene family, wherein the genes orgene family sequences share sequence homology. Such homologous sequencescan be identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering PGF-1 targets that share sequence homology. As such, oneadvantage of using siNAs of the invention is that a single siNA can bedesigned to include nucleic acid sequence that is complementary to thenucleotide sequence that is conserved between the homologous genes. Inthis approach, a single siNA can be used to inhibit expression of morethan one gene instead of using more than one siNA molecule to target thedifferent genes.

In one embodiment, the invention features an siNA molecule having RNAiactivity against PGF-1 RNA, wherein the siNA molecule comprises asequence complementary to any RNA having PGF-1 encoding sequence, suchas those sequences having GenBank Accession Nos. shown in Table I. Inanother embodiment, the invention features an siNA molecule having RNAiactivity against PGF-1 RNA, wherein the siNA molecule comprises asequence complementary to an RNA having variant PGF-1 encoding sequence,for example other mutant PGF-1 genes not shown in Table I but known inthe art to be associated with the maintenance and/or development ofcancer, proliferative diseases, disorders, or conditions, oculardiseases, disorders, or conditions, dermatological diseases, disorders,or conditions, rheumatoid arthritis, endometriosis, and kidney disease.Chemical modifications as shown in Tables III and IV or otherwisedescribed herein can be applied to any siNA construct of the invention.In another embodiment, an siNA molecule of the invention includes anucleotide sequence that can interact with nucleotide sequence of aPGF-1 gene and thereby mediate silencing of PGF-1 gene expression, forexample, wherein the siNA mediates regulation of PGF-1 gene expressionby cellular processes that modulate the chromatin structure ormethylation patterns of the PGF-1 gene and prevent transcription of thePGF-1 gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of PGF-1 proteins arising from PGF-1haplotype polymorphisms that are associated with a disease or condition,(e.g., cancer, proliferative diseases, disorders, or conditions, oculardiseases, disorders, or conditions, dermatological diseases, disorders,or conditions, rheumatoid arthritis, endometriosis, and kidney disease).Analysis of PGF-1 genes, or PGF-1 protein or RNA levels can be used toidentify subjects with such polymorphisms or those subjects who are atrisk of developing traits, conditions, or diseases described herein.These subjects are amenable to treatment, for example, treatment withsiNA molecules of the invention and any other composition useful intreating diseases related to PGF-1 gene expression. As such, analysis ofPGF-1 protein or RNA levels can be used to determine treatment type andthe course of therapy in treating a subject. Monitoring of PGF-1 proteinor RNA levels can be used to predict treatment outcome and to determinethe efficacy of compounds and compositions that modulate the leveland/or activity of certain PGF-1 proteins associated with a trait,condition, or disease.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a PGF-1 protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a PGF-1 gene or a portion thereof.

In another embodiment, an siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a PGF-1 protein or a portion thereof. The siNAmolecule further comprises a sense region, wherein said sense regioncomprises a nucleotide sequence of a PGF-1 gene or a portion thereof.

In another embodiment, the invention features an siNA moleculecomprising a nucleotide sequence in the antisense region of the siNAmolecule that is complementary to a nucleotide sequence or portion ofsequence of a PGF-1 gene. In another embodiment, the invention featuresan siNA molecule comprising a region, for example, the antisense regionof the siNA construct, complementary to a sequence comprising a PGF-1gene sequence or a portion thereof.

In one embodiment, the antisense region of PGF-1 siNA constructscomprises a sequence complementary to sequence having any of SEQ ID NOs.1-97 or 195-202. In one embodiment, the antisense region of PGF-1constructs comprises sequence having any of SEQ ID NOs. 98-194, 211-218,227-234, 243-250, 259-266, 275-298, 300, 302, 304, 307, 309, 311, 313,or 316. In another embodiment, the sense region of PGF-1 constructscomprises sequence having any of SEQ ID NOs. 1-97, 195-210, 219-226,235-242, 251-258, 267-274, 299, 301, 303, 305, 306, 308, 310, 312, 314,or 315.

In one embodiment, an siNA molecule of the invention comprises any ofSEQ ID NOs. 1-316. The sequences shown in SEQ ID NOs: 1-316 are notlimiting. An siNA molecule of the invention can comprise any contiguousPGF-1 sequence (e.g., about 15 to about 25 or more, or about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous PGF-1 nucleotides).

In yet another embodiment, the invention features an siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a PGF-1 protein, and wherein said siNA furthercomprises a sense strand having about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences where at least about 15 nucleotides ineach strand are complementary to the other strand.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a RNAsequence encoding a PGF-1 protein, and wherein said siNA furthercomprises a sense region having about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein said sense region and said antisense region arecomprised in a linear molecule where the sense region comprises at leastabout 15 nucleotides that are complementary to the antisense region.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a PGF-1 gene. Becauseplacental growth factor genes (e.g., PGF-1, PGF-2, and/or PGF-3) genescan share some degree of sequence homology with each other, siNAmolecules can be designed to target a class of placental growth factorgenes (and associated receptor or ligand genes) or alternately specificplacental growth factor genes (e.g., polymorphic variants) by selectingsequences that are either shared amongst different placental growthfactor targets or alternatively that are unique for a specific placentalgrowth factor target. Therefore, in one embodiment, the siNA moleculecan be designed to target conserved regions of PGF-1 RNA sequenceshaving homology among several PGF-1 gene variants so as to target aclass of PGF-1 genes (e.g., PGF-1, different PGF-1 isoforms such asPGF-2 and PGF-3, splice variants, mutant genes etc.) with one siNAmolecule. Accordingly, in one embodiment, the siNA molecule of theinvention modulates the expression of one or both PGF-1 alleles in asubject. In another embodiment, the siNA molecule can be designed totarget a sequence that is unique to a specific PGF-1 RNA sequence (e.g.,a single PGF-1 allele or PGF-1 single nucleotide polymorphism (SNP)) dueto the high degree of specificity that the siNA molecule requires tomediate RNAi activity.

In one embodiment, an siNA molecule of the invention targeting PGF-1 isused in combination with another anti-angiogenic therapy or compound.Such anti-angiogenic therapies and compounds can include compositionsthat modulate the expression or activity of vascular endothelial growthfactor (VEGF) and/or its receptors (e.g. VEGFR1, VEGFR2, and/or VEGFR3).For example, an siNA molecule of the invention targeting PGF-1 is usedin combination with one or more siNA molecules targeting VEGF or VEGFreceptors (e.g. VEGFR1, VEGFR2, and/or VEGFR3), as described in Pavco,U.S. Ser. No. 60/334,461, filed Nov. 3, 2001; Pavco, International PCTPublication No. WO 02/096927, filed May 29, 2002, McSwiggen et al., U.S.Ser. No. 10/670,011, filed Sep. 23, 2003, McSwiggen et al., U.S. Ser.No. 10/664,668, filed Sep. 18, 2003, and McSwiggen et al., U.S. Ser. No.10/665,951, filed Sep. 18, 2003, all incorporated by reference herein intheir entirety including the drawings.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 15 to about 30 base pairs between oligonucleotidescomprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2,or 3) nucleotides, for example, about 21-nucleotide duplexes with about19 base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs. In yet another embodiment, siNA molecules ofthe invention comprise duplex nucleic acid molecules with blunt ends,where both ends are blunt, or alternatively, where one of the ends isblunt.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for PGF-1expressing nucleic acid molecules, such as RNA encoding a PGF-1 protein.In one embodiment, the invention features a RNA based siNA molecule(e.g., an siNA comprising 2′-OH nucleotides) having specificity forPGF-1 expressing nucleic acid molecules that includes one or morechemical modifications described herein. Non-limiting examples of suchchemical modifications include without limitation phosphorothioateinternucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminalglyceryl and/or inverted deoxy abasic residue incorporation. Thesechemical modifications, when used in various siNA constructs, (e.g., RNAbased siNA constructs), are shown to preserve RNAi activity in cellswhile at the same time, dramatically increasing the serum stability ofthese compounds. Furthermore, contrary to the data published by Parrishet al., supra, applicant demonstrates that multiple (greater than one)phosphorothioate substitutions are well-tolerated and confer substantialincreases in serum stability for modified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, ansiNA molecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, an siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single-stranded, the percent modification can be based uponthe total number of nucleotides present in the single-stranded siNAmolecules. Likewise, if the siNA molecule is double-stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a PGF-1gene. In one embodiment, the double-stranded siNA molecule comprises oneor more chemical modifications and each strand of the double-strandedsiNA is about 21 nucleotides long. In one embodiment, thedouble-stranded siNA molecule does not contain any ribonucleotides. Inanother embodiment, the double-stranded siNA molecule comprises one ormore ribonucleotides. In one embodiment, each strand of thedouble-stranded siNA molecule independently comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides, wherein each strand comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides that are complementary to the nucleotides of theother strand. In one embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence or a portion thereof of the PGF-1gene, and the second strand of the double-stranded siNA moleculecomprises a nucleotide sequence substantially similar to the nucleotidesequence of the PGF-1 gene or a portion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene comprising an antisense region, wherein the antisenseregion comprises a nucleotide sequence that is complementary to anucleotide sequence of the PGF-1 gene or a portion thereof, and a senseregion, wherein the sense region comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the PGF-1 gene or aportion thereof. In one embodiment, the antisense region and the senseregion independently comprise about 15 to about 30 (e.g. about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense region comprises about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the PGF-1 geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region.

In one embodiment, an siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, an siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 32” (Table IV) or any combination thereof(see Table IV)) and/or any length described herein can comprise bluntends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, an siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of adouble-stranded siNA molecule having no overhanging nucleotides. The twostrands of a double-stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. The sense region can be connected to the antisenseregion via a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene, wherein the siNA molecule comprises about 15 to about30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22; 23, 24, 25, 26, 27, 28,29, or 30) base pairs, and wherein each strand of the siNA moleculecomprises one or more chemical modifications. In another embodiment, oneof the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of aPGF-1 gene or a portion thereof, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or a portion thereof ofthe PGF-1 gene. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a PGF-1 gene or portionthereof, and the second strand of the double-stranded siNA moleculecomprises a nucleotide sequence substantially similar to the nucleotidesequence or portion thereof of the PGF-1 gene. In another embodiment,each strand of the siNA molecule comprises about 15 to about 30 (e.g.about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and each strand comprises at least about 15 to about 30(e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30) nucleotides that are complementary to the nucleotides of theother strand. The PGF-1 gene can comprise, for example, sequencesreferred to in Table I.

In one embodiment, an siNA molecule of the invention comprises noribonucleotides. In another embodiment, an siNA molecule of theinvention comprises ribonucleotides.

In one embodiment, an siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a PGF-1 gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the PGF-1 gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.The PGF-1 gene can comprise, for example, sequences referred to in TableI. In another embodiment, the siNA is a double-stranded nucleic acidmolecule, where each of the two strands of the siNA moleculeindependently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36,37, 38, 39, or 40) nucleotides, and where one of the strands of the siNAmolecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20,21, 22, 23, 24 or 25 or more) nucleotides that are complementary to thenucleic acid sequence of the PGF-1 gene or a portion thereof.

In one embodiment, an siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a PGF-1 gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The PGF-1 genecan comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the PGF-1 geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thesiNA molecule has one or more modified pyrimidine and/or purinenucleotides. In one embodiment, the pyrimidine nucleotides in the senseregion are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-O-methyl purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In one embodiment, thepyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in theantisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. Inanother embodiment of any of the above-described siNA molecules, anynucleotides present in a non-complementary region of the sense strand(e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule, and wherein the fragment comprising the sense regionincludes a terminal cap moiety at the 5′-end, the 3′-end, or both of the5′ and 3′ ends of the fragment. In one embodiment, the terminal capmoiety is an inverted deoxy abasic moiety or glyceryl moiety. In oneembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In anotherembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39,or 40) nucleotides. In a non-limiting example, each of the two fragmentsof the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features an siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, about 15 toabout 40 nucleotides in length. In one embodiment, all pyrimidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidinenucleotides. In one embodiment, the modified nucleotides in the siNAinclude at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluorouridine nucleotide. In another embodiment, the modified nucleotides inthe siNA include at least one 2′-deoxy-2′-fluoro cytidine and at leastone 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, alluridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing thestability of an siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-fluoro-2′-deoxy cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the PGF-1 geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thepurine nucleotides present in the antisense region comprise2′-deoxy-purine nucleotides. In an alternative embodiment, the purinenucleotides present in the antisense region comprise 2′-O-methyl purinenucleotides. In either of the above embodiments, the antisense regioncan comprise a phosphorothioate internucleotide linkage at the 3′ end ofthe antisense region. Alternatively, in either of the above embodiments,the antisense region can comprise a glyceryl modification at the 3′ endof the antisense region. In another embodiment of any of theabove-described siNA molecules, any nucleotides present in anon-complementary region of the antisense strand (e.g. overhang region)are 2′-deoxy nucleotides.

In one embodiment, the antisense region of an siNA molecule of theinvention comprises sequence complementary to a portion of a PGF-1transcript having sequence unique to a particular PGF-1 disease relatedallele, such as sequence comprising a single nucleotide polymorphism(SNP) associated with the disease specific allele. As such, theantisense region of an siNA molecule of the invention can comprisesequence complementary to sequences that are unique to a particularallele to provide specificity in mediating selective RNAi against thedisease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a PGF-1 gene, wherein the siNA molecule is assembled from twoseparate oligonucleotide fragments wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the siNA molecule is adouble-stranded nucleic acid molecule, where each strand is about 21nucleotides long and where about 19 nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule, wherein at least two 3′ terminalnucleotides of each fragment of the siNA molecule are not base-paired tothe nucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double-stranded nucleic acidmolecule, where each strand is about 19 nucleotide long and where thenucleotides of each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule toform at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, whereinone or both ends of the siNA molecule are blunt ends. In one embodiment,each of the two 3′ terminal nucleotides of each fragment of the siNAmolecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double-stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region, where about 19 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the PGF-1 gene. In another embodiment, about 21 nucleotidesof the antisense region are base-paired to the nucleotide sequence or aportion thereof of the RNA encoded by the PGF-1 gene. In any of theabove embodiments, the 5′-end of the fragment comprising said antisenseregion can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa PGF-1 RNA sequence (e.g., wherein said target RNA sequence is encodedby a PGF-1 gene involved in the PGF-1 pathway), wherein the siNAmolecule does not contain any ribonucleotides and wherein each strand ofthe double-stranded siNA molecule is about 15 to about 30 nucleotides.In one embodiment, the siNA molecule is 21 nucleotides in length.Examples of non-ribonucleotide containing siNA constructs arecombinations of stabilization chemistries shown in Table IV in anycombination of Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11,Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12,13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or anycombination thereof).

In one embodiment, the invention features a chemically synthesizeddouble-stranded RNA molecule that directs cleavage of a PGF-1 RNA viaRNA interference, wherein each strand of said RNA molecule is about 15to about 30 nucleotides in length; one strand of the RNA moleculecomprises nucleotide sequence having sufficient complementarity to thePGF-1 RNA for the RNA molecule to direct cleavage of the PGF-1 RNA viaRNA interference; and wherein at least one strand of the RNA moleculeoptionally comprises one or more chemically modified nucleotidesdescribed herein, such as without limitation deoxynucleotides,2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides,2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising ansiNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a PGF-1 gene, wherein the siNAmolecule comprises one or more chemical modifications and each strand ofthe double-stranded siNA is independently about 15 to about 30 or more(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 or more) nucleotides long. In one embodiment, the siNA molecule ofthe invention is a double-stranded nucleic acid molecule comprising oneor more chemical modifications, where each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of thestrands comprises at least 15 nucleotides that are complementary tonucleotide sequence of PGF-1 encoding RNA or a portion thereof. In anon-limiting example, each of the two fragments of the siNA moleculecomprise about 21 nucleotides. In another embodiment, the siNA moleculeis a double-stranded nucleic acid molecule comprising one or morechemical modifications, where each strand is about 21 nucleotide longand where about 19 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule, wherein at least two 3′ terminal nucleotides of eachfragment of the siNA molecule are not base-paired to the nucleotides ofthe other fragment of the siNA molecule. In another embodiment, the siNAmolecule is a double-stranded nucleic acid molecule comprising one ormore chemical modifications, where each strand is about 19 nucleotidelong and where the nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or19) base pairs, wherein one or both ends of the siNA molecule are bluntends. In one embodiment, each of the two 3′ terminal nucleotides of eachfragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, suchas a 2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double-stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region and comprising one or more chemical modifications,where about 19 nucleotides of the antisense region are base-paired tothe nucleotide sequence or a portion thereof of the RNA encoded by thePGF-1 gene. In another embodiment, about 21 nucleotides of the antisenseregion are base-paired to the nucleotide sequence or a portion thereofof the RNA encoded by the PGF-1 gene. In any of the above embodiments,the 5′-end of the fragment comprising said antisense region canoptionally include a phosphate group.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits,down-regulates, or reduces expression of a PGF-1 gene, wherein one ofthe strands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of PGF-1 RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a PGF-1 gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofPGF-1 RNA or a portion thereof, wherein the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a PGF-1 gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence ofPGF-1 RNA that encodes a protein or portion thereof, the other strand isa sense strand which comprises nucleotide sequence that is complementaryto a nucleotide sequence of the antisense strand and wherein a majorityof the pyrimidine nucleotides present in the double-stranded siNAmolecule comprises a sugar modification. In one embodiment, each strandof the siNA molecule comprises about 15 to about 30 or more (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ormore) nucleotides, wherein each strand comprises at least about 15nucleotides that are complementary to the nucleotides of the otherstrand. In one embodiment, the siNA molecule is assembled from twooligonucleotide fragments, wherein one fragment comprises the nucleotidesequence of the antisense strand of the siNA molecule and a secondfragment comprises nucleotide sequence of the sense region of the siNAmolecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPGF-1 gene, wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, eachof the two strands of the siNA molecule can comprise about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule. In another embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In one embodiment, each strand of the siNA molecule is base-paired tothe complementary nucleotides of the other strand of the siNA molecule.In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of theantisense strand are base-paired to the nucleotide sequence of the PGF-1RNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g.,about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisensestrand are base-paired to the nucleotide sequence of the PGF-1 RNA or aportion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPGF-1 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of PGF-1 RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the 5′-end of the antisense strand optionally includes aphosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPGF-1 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of PGF-1 RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the untranslatedregion or a portion thereof of the PGF-1 RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aPGF-1 gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of PGF-1 RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand, wherein a majority of the pyrimidine nucleotidespresent in the double-stranded siNA molecule comprises a sugarmodification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the PGF-1 RNA or aportion thereof that is present in the PGF-1 RNA.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of an siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of an siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding PGF-1 and thesense region can comprise sequence complementary to the antisenseregion. The siNA molecule can comprise two distinct strands havingcomplementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PGF-1 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotidescomprising a backbone modified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl andwherein W, X, Y, and Z are optionally not all O. In another embodiment,a backbone modification of the invention comprises a phosphonoacetateand/or thiophosphonoacetate internucleotide linkage (see for exampleSheehan et al., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, an siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PGF-1 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PGF-1 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or H; R9 isO, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, an siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PGF-1 inside a cell or reconstituted invitro system, wherein the chemical modification comprises a 5′-terminalphosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all 0.

In one embodiment, the invention features an siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features an siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of an siNA molecule of the invention,for example an siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against PGF-1 inside a cell or reconstituted invitro system, wherein the chemical modification comprises one or morephosphorothioate internucleotide linkages. For example, in anon-limiting example, the invention features a chemically-modified shortinterfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 ormore phosphorothioate internucleotide linkages in one siNA strand. Inyet another embodiment, the invention features a chemically-modifiedshort interfering nucleic acid (siNA) individually having about 1, 2, 3,4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in bothsiNA strands. The phosphorothioate internucleotide linkages can bepresent in one or both oligonucleotide strands of the siNA duplex, forexample in the sense strand, the antisense strand, or both strands. ThesiNA molecules of the invention can comprise one or morephosphorothioate internucleotide linkages at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the sense strand, the antisense strand,or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) consecutive phosphorothioate internucleotide linkages atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine phosphorothioate internucleotide linkages inthe sense strand, the antisense strand, or both strands. In yet anothernon-limiting example, an exemplary siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) purine phosphorothioate internucleotide linkages in the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of one or both siNA sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both siNA sequence strands, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in one or both strands of the siNA molecule cancomprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more including every internucleotide linkage of a purinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, an siNA molecule of the invention comprises asingle-stranded hairpin structure, wherein the siNA is about 36 to about70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification comprising a structure having any ofFormulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically-modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric double-stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25; 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprisesan asymmetric double-stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double-stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2, nucleotides.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3,R8 or R13 serves as a point of attachment to the siNA molecule of theinvention.

In another embodiment, an siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for example, seemodification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of theinvention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends ofan siNA molecule of the invention. For example, chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) can be present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense strand, the sense strand, or both antisense andsense strands of the siNA molecule. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the 5′-end and 3′-end of the sense strand andthe 3′-end of the antisense strand of a double-stranded siNA molecule ofthe invention. In one embodiment, the chemically modified nucleoside ornon-nucleoside (e.g., a moiety having Formula V, VI or VII) is presentat the terminal position of the 5′-end and 3′-end of the sense strandand the 3′-end of the antisense strand of a double-stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the two terminal positions of the 5′-end and 3′-endof the sense strand and the 3′-end of the antisense strand of adouble-stranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double-stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a 3′-3′, 3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against PGF-1 inside a cell orreconstituted in vitro system comprising a sense region, wherein one ormore pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), and an antisenseregion, wherein one or more pyrimidine nucleotides present in theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modificationdescribed herein or shown in FIG. 10, that is optionally present at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/orantisense sequence. The sense and/or antisense region can optionallyfurther comprise a 3′-terminal nucleotide overhang having about 1 toabout 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. Non-limiting examples of thesechemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III andIV herein. In any of these described embodiments, the purine nucleotidespresent in the sense region are alternatively 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides) and one or more purine nucleotidespresent in the antisense region are 2′-O-methyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotidesor alternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). Also, in any of these embodiments, one or more purinenucleotides present in the sense region are alternatively purineribonucleotides (e.g., wherein all purine nucleotides are purineribonucleotides or alternately a plurality of purine nucleotides arepurine ribonucleotides) and any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double-stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against PGF-1 inside a cell or reconstituted invitro system, wherein the chemical modification comprises a conjugatecovalently attached to the chemically-modified siNA molecule.Non-limiting examples of conjugates contemplated by the inventioninclude conjugates and ligands described in Vargeese et al., U.S. Ser.No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein inits entirety, including the drawings. In another embodiment, theconjugate is covalently attached to the chemically-modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically-modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically-modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically-modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically-modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has a sequence that comprises a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, an siNA molecule canbe assembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, an siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence.In another embodiment, the single-stranded siNA molecule of theinvention comprises a 5′-terminal phosphate group. In anotherembodiment, the single-stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a2′,3′-cyclic phosphate). In another embodiment, the single-stranded siNAmolecule of the invention comprises about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In yet another embodiment, the single-stranded siNAmolecule of the invention comprises one or more chemically modifiednucleotides or non-nucleotides described herein. For example, all thepositions within the siNA molecule can include chemically-modifiednucleotides such as nucleotides having any of Formulae I-VII, or anycombination thereof to the extent that the ability of the siNA moleculeto support RNAi activity in a cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single-strandedpolynucleotide having complementarity to a target nucleic acid sequence,wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 10, that is optionally present at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the antisense sequence. ThesiNA optionally further comprises about 1 to about 4 or more (e.g.,about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end ofthe siNA molecule, wherein the terminal nucleotides can further compriseone or more (e.g., 1, 2, 3, 4 or more) phosphorothioate,phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages,and wherein the siNA optionally further comprises a terminal phosphategroup, such as a 5′-terminal phosphate group. In any of theseembodiments, any purine nucleotides present in the antisense region arealternatively 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides). Also, in any ofthese embodiments, any purine nucleotides present in the siNA (i.e.,purine nucleotides present in the sense and/or antisense region) canalternatively be locked nucleic acid (LNA) nucleotides (e.g., whereinall purine nucleotides are LNA nucleotides or alternately a plurality ofpurine nucleotides are LNA nucleotides). Also, in any of theseembodiments, any purine nucleotides present in the siNA arealternatively 2′-methoxyethyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-methoxyethyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides). In another embodiment, any modified nucleotides present inthe single-stranded siNA molecules of the invention comprise modifiednucleotides having properties or characteristics similar to naturallyoccurring ribonucleotides. For example, the invention features siNAmolecules including modified nucleotides having a Northern conformation(e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Assuch, chemically modified nucleotides present in the single-strandedsiNA molecules of the invention are preferably resistant to nucleasedegradation while at the same time maintaining the capacity to mediateRNAi.

In one embodiment, an siNA molecule of the invention compriseschemically modified nucleotides or non-nucleotides (e.g., having any ofFormulae I-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides) at alternating positions within one or more strands orregions of the siNA molecule. For example, such chemical modificationscan be introduced at every other position of a RNA based siNA molecule,starting at either the first or second nucleotide from the 3′-end or5′-end of the siNA. In a non-limiting example, a double-stranded siNAmolecule of the invention in which each strand of the siNA is 21nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11,13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., withcompounds having any of Formulae I-VI, such as such as 2′-deoxy,2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides). In another non-limitingexample, a double-stranded siNA molecule of the invention in which eachstrand of the siNA is 21 nucleotides in length is featured whereinpositions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand arechemically modified (e.g., with compounds having any of Formulae I-VII,such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides). Such siNA molecules can further comprise terminal capmoieties and/or backbone modifications as described herein.

In one embodiment, the invention features a method for modulating theexpression of a PGF-1 gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically-modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the PGF-1 gene; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the PGF-1 genein the cell.

In one embodiment, the invention features a method for modulating theexpression of a PGF-1 gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically-modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the PGF-1 gene and wherein the sense strand sequence of the siNAcomprises a sequence identical or substantially similar to the sequenceof the target RNA; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the PGF-1 genein the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one PGF-1 gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PGF-1 genes; and (b) introducingthe siNA molecules into a cell under conditions suitable to modulate theexpression of the PGF-1 genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more PGF-1 genes within a cell comprising: (a)synthesizing one or more siNA molecules of the invention, which can bechemically-modified, wherein the siNA strands comprise sequencescomplementary to RNA of the PGF-1 genes and wherein the sense strandsequences of the siNAs comprise sequences identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecules into a cell under conditions suitable to modulate theexpression of the PGF-1 genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one PGF-1 gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PGF-1 gene and wherein the sensestrand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the PGF-1 genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of a PGF-1 genein a tissue explant comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the PGF-1 gene; and(b) introducing the siNA molecule into a cell of the tissue explantderived from a particular organism under conditions suitable to modulatethe expression of the PGF-1 gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of thePGF-1 gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a PGF-1 gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PGF-1 gene and wherein the sensestrand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the target RNA; and (b)introducing the siNA molecule into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the PGF-1 gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of thePGF-1 gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PGF-1 gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PGF-1 genes; and (b) introducingthe siNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the PGF-1 genes in the tissue explant. In another embodiment, themethod further comprises introducing the tissue explant back into theorganism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the PGF-1 genes inthat organism.

In one embodiment, the invention features a method of modulating theexpression of a PGF-1 gene in a subject or organism comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PGF-1 gene; and (b) introducing thesiNA molecule into the subject or organism under conditions suitable tomodulate the expression of the PGF-1 gene in the subject or organism.The level of PGF-1 protein or RNA can be determined using variousmethods well-known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one PGF-1 gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the PGF-1 genes; and (b) introducingthe siNA molecules into the subject or organism under conditionssuitable to modulate the expression of the PGF-1 genes in the subject ororganism. The level of PGF-1 protein or RNA can be determined as isknown in the art.

In one embodiment, the invention features a method for modulating theexpression of a PGF-1 gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically-modified,wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the PGF-1 gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate theexpression of the PGF-1 gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one PGF-1 gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the PGF-1 gene; and (b)contacting the cell in vitro or in vivo with the siNA molecule underconditions suitable to modulate the expression of the PGF-1 genes in thecell.

In one embodiment, the invention features a method of modulating theexpression of a PGF-1 gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the PGF-1 gene; and (b)contacting a cell of the tissue explant derived from a particularsubject or organism with the siNA molecule under conditions suitable tomodulate the expression of the PGF-1 gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the subject or organism the tissue was derived from orinto another subject or organism under conditions suitable to modulatethe expression of the PGF-1 gene in that subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PGF-1 gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the PGF-1 gene; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular subject or organism under conditions suitable tomodulate the expression of the PGF-1 genes in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the subject or organism the tissue was derived from orinto another subject or organism under conditions suitable to modulatethe expression of the PGF-1 genes in that subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a PGF-1 gene in a subject or organism comprising: (a)synthesizing an siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the PGF-1 gene; and (b)introducing the siNA molecule into the subject or organism underconditions suitable to modulate the expression of the PGF-1 gene in thesubject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PGF-1 gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the PGF-1 gene; and (b)introducing the siNA molecules into the subject or organism underconditions suitable to modulate the expression of the PGF-1 genes in thesubject or organism.

In one embodiment, the invention features a method of modulating theexpression of a PGF-1 gene in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of thePGF-1 gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing cancer in a subject or organism comprising contacting thesubject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the PGF-1 gene in thesubject or organism.

In one embodiment, the invention features a method for treating orpreventing a proliferative disease, disorder, or condition in a subjector organism comprising contacting the subject or organism with an siNAmolecule of the invention under conditions suitable to modulate theexpression of the PGF-1 gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing an ocular disease, disorder, or condition in a subject ororganism comprising contacting the subject or organism with an siNAmolecule of the invention under conditions suitable to modulate theexpression of the PGF-1 gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a dermatological disease, disorder, or condition in a subjector organism comprising contacting the subject or organism with an siNAmolecule of the invention under conditions suitable to modulate theexpression of the PGF-1 gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing rheumatoid arthritis in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of thePGF-1 gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing endometriosis in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the PGF-1 gene in thesubject or organism.

In one embodiment, the invention features a method for treating orpreventing kidney disease in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the PGF-1 gene in thesubject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one PGF-1 genes in a subject or organismcomprising contacting the subject or organism with one or more siNAmolecules of the invention under conditions suitable to modulate theexpression of the PGF-1 genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., PGF-1) gene expression through RNAi targeting of avariety of RNA molecules. In one embodiment, the siNA molecules of theinvention are used to target various RNAs corresponding to a targetgene. Non-limiting examples of such RNAs include messenger RNA (mRNA),alternate RNA splice variants of target gene(s), post-transcriptionallymodified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNAtemplates. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as PGF-1 family genes. As such, siNA molecules targetingmultiple PGF-1 targets can provide increased therapeutic effect. Inaddition, siNA can be used to characterize pathways of gene function ina variety of applications. For example, the present invention can beused to inhibit the activity of target gene(s) in a pathway to determinethe function of uncharacterized gene(s) in gene function analysis, mRNAfunction analysis, or translational analysis. The invention can be usedto determine potential target gene pathways involved in various diseasesand conditions toward pharmaceutical development. The invention can beused to understand pathways of gene expression involved in, for examplecancer, proliferative diseases, disorders, or conditions, oculardiseases, disorders, or conditions, dermatological diseases, disorders,or conditions, rheumatoid arthritis, endometriosis, and kidney disease.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession No., for example, PGF-1 genes encoding RNAsequence(s) referred to herein by Genbank Accession number, for example,Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described herein. In another embodiment, the assaycan comprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of target RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(e.g., for an siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target PGF-1 RNA sequence. Inanother embodiment, the siNA molecules of (a) have strands of a fixedlength, for example about 23 nucleotides in length. In yet anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described in Example 6 herein. In anotherembodiment, the assay can comprise a cell culture system in which targetRNA is expressed. In another embodiment, fragments of PGF-1 RNA areanalyzed for detectable levels of cleavage, for example, by gelelectrophoresis, northern blot analysis, or RNAse protection assays, todetermine the most suitable target site(s) within the target PGF-1 RNAsequence. The target PGF-1 RNA sequence can be obtained as is known inthe art, for example, by cloning and/or transcription for in vitrosystems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides in length. In one embodiment, the assay cancomprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. Fragments of target RNA are analyzed fordetectable levels of cleavage, for example by gel electrophoresis,northern blot analysis, or RNAse protection assays, to determine themost suitable target site(s) within the target RNA sequence. The targetRNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by expression inin vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by an siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention, which can be chemically-modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically-modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for treating orpreventing cancer, proliferative diseases, disorders, or conditions,ocular diseases, disorders, or conditions, dermatological diseases,disorders, or conditions, rheumatoid arthritis, endometriosis, andkidney disease in a subject or organism comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of cancer, proliferative diseases, disorders, orconditions, ocular diseases, disorders, or conditions, dermatologicaldiseases, disorders, or conditions, rheumatoid arthritis, endometriosis,and kidney disease in the subject or organism.

In another embodiment, the invention features a method for validating aPGF-1 gene target, comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a PGF-1 target gene;(b) introducing the siNA molecule into a cell, tissue, subject, ororganism under conditions suitable for modulating expression of thePGF-1 target gene in the cell, tissue, subject, or organism; and (c)determining the function of the gene by assaying for any phenotypicchange in the cell, tissue, subject, or organism.

In another embodiment, the invention features a method for validating aPGF-1 target comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a PGF-1 target gene;(b) introducing the siNA molecule into a biological system underconditions suitable for modulating expression of the PGF-1 target genein the biological system; and (c) determining the function of the geneby assaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, subject, or organism, or extract thereof. The term biologicalsystem also includes reconstituted RNAi systems that can be used in anin vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing an siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a PGF-1 target gene in a biologicalsystem, including, for example, in a cell, tissue, subject, or organism.In another embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically-modified,that can be used to modulate the expression of more than one PGF-1target gene in a biological system, including, for example, in a cell,tissue, subject, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing an siNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing ansiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizingan siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against PGF-1, wherein the siNA construct comprises one or morechemical modifications, for example, one or more chemical modificationshaving any of Formulae I-VII or any combination thereof that increasesthe nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In another embodiment, the invention features a method for generatingsiNA molecules with improved toxicologic profiles (e.g., have attenuatedor no immunostimulatory properties) comprising (a) introducingnucleotides having any of Formula I-VII (e.g., siNA motifs referred toin Table IV) or any combination thereof into an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) introducing nucleotides having anyof Formula I-VII (e.g., siNA motifs referred to in Table IV) or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate an interferon response.

By “improved toxicologic profile”, is meant that the chemically modifiedsiNA construct exhibits decreased toxicity in a cell, subject, ororganism compared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In a non-limiting example, siNA molecules withimproved toxicologic profiles are associated with a decreased orattenuated immunostimulatory response in a cell, subject, or organismcompared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In one embodiment, an siNA molecule with animproved toxicological profile comprises no ribonucleotides. In oneembodiment, an siNA molecule with an improved toxicological profilecomprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4ribonucleotides). In one embodiment, an siNA molecule with an improvedtoxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab25, Stab 26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32 or anycombination thereof (see Table IV). In one embodiment, the level ofimmunostimulatory response associated with a given siNA molecule can bemeasured as is known in the art, for example by determining the level ofPKR/interferon response, proliferation, B-cell activation, and/orcytokine production in assays to quantitate the immunostimulatoryresponse of particular siNA molecules (see, for example, Leifer et al.,2003, J. Immunother. 26, 313-9; and U.S. Pat. No. 5,968,909,incorporated in its entirety by reference).

In one embodiment, the invention features siNA constructs that mediateRNAi against PGF-1, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoan siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against PGF-1, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against PGF-1, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against PGF-1, wherein the siNA construct comprises one or morechemical modifications described herein that modulate the polymeraseactivity of a cellular polymerase capable of generating additionalendogenous siNA molecules having sequence homology to thechemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule.

In one embodiment, the invention features chemically-modified siNAconstructs that mediate RNAi against PGF-1 in a cell, wherein thechemical modifications do not significantly effect the interaction ofsiNA with a target RNA molecule, DNA molecule and/or proteins or otherfactors that are essential for RNAi in a manner that would decrease theefficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against PGF-1 comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against PGF-1target RNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against PGF-1target DNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against PGF-1, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the cellularuptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against PGF-1 with improved cellular uptake comprising(a) introducing nucleotides having any of Formula I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against PGF-1, wherein the siNA construct comprises one or morechemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of an siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, an siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (seeTable IV) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of an siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”,“Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23,or 24 sense strands) chemistries and variants thereof (see Table IV)wherein the 5′-end and 3′-end of the sense strand of the siNA do notcomprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include an siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Tables IIand III herein. For example the siNA can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex ordouble-stranded structure, for example wherein the double-strandedregion is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strandcomprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense strand comprises nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof (e.g., about 15 to about 25or more nucleotides of the siNA molecule are complementary to the targetnucleic acid or a portion thereof). Alternatively, the siNA is assembledfrom a single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s). The siNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single-stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single-stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy(2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level and the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

In one embodiment, an siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. US04/16390, filed May 24, 2004).

In one embodiment, an siNA molecule of the invention is amultifunctional siNA, (see for example FIGS. 16-21 and Jadhav et al.,U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCTApplication No. US04/16390, filed May 24, 2004). The multifunctionalsiNA of the invention can comprise sequence targeting, for example, tworegions of PGF-1 RNA (see for example target sequences in Tables II andIII).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant an siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing.

By “gene”, or “target gene”, is meant a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Aberrant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of asubject, organism or cell, by intervening in cellular processes such asgenetic imprinting, transcription, translation, or nucleic acidprocessing (e.g., transamination, methylation etc.). The target gene canbe a gene derived from a cell, an endogenous gene, a transgene, orexogenous genes such as genes of a pathogen, for example a virus, whichis present in the cell after infection thereof. The cell containing thetarget gene can be derived from or contained in any organism, forexample a plant, animal, protozoan, virus, bacterium, or fungus.Non-limiting examples of plants include monocots, dicots, orgymnosperms. Non-limiting examples of animals include vertebrates orinvertebrates. Non-limiting examples of fungi include molds or yeasts.For a review, see for example Snyder and Gerstein, 2003, Science, 300,258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N-3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “PGF-1” as used herein is meant, any placental growth factor (e.g.,PGF-1) protein, peptide, or polypeptide having any placental growthfactor activity, such as encoded by placental growth factor GenbankAccession Nos. shown in Table I. The term PGF-1 also refers to nucleicacid sequences encoding any placental growth factor protein, peptide, orpolypeptide having placental growth factor activity. The term “PGF-1” isalso meant to include other PGF-1 encoding sequence, such as otherplacental growth factor isoforms (such as PGF-2 and PGF-3), mutant PGF-1genes, splice variants of PGF-1 genes, and PGF-1 gene polymorphisms.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of an siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of an siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, an siNA moleculeof the invention comprises about 15 to about 30 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)nucleotides that are complementary to one or more target nucleic acidmolecules or a portion thereof.

In one embodiment, siNA molecules of the invention that down regulate orreduce PGF-1 gene expression are used for preventing or treating cancer,proliferative diseases, disorders, or conditions, ocular diseases,disorders, or conditions, dermatological diseases, disorders, orconditions, rheumatoid arthritis, endometriosis, and kidney disease in asubject or organism.

In one embodiment, the siNA molecules of the invention are used to treatcancer, proliferative diseases, disorders, or conditions, oculardiseases, disorders, or conditions, dermatological diseases, disorders,or conditions, rheumatoid arthritis, endometriosis, and kidney disease,disorders, and/or conditions in a subject or organism.

By “proliferative disease” or “cancer” as used herein is meant, anydisease, condition, trait, genotype or phenotype characterized byunregulated cell growth or replication as is known in the art; includingleukemias, for example, acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia, AIDS related cancers such as Kaposi'ssarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “ocular disease” as used herein is meant, any disease, condition,trait, genotype or phenotype of the eye and related structures, such asCystoid Macular Edema, Asteroid Hyalosis, Pathological Myopia andPosterior Staphyloma, Toxocariasis (Ocular Larva Migrans), Retinal VeinOcclusion, Posterior Vitreous Detachment, Tractional Retinal Tears,Epiretinal Membrane, Diabetic Retinopathy, Lattice Degeneration, RetinalVein Occlusion, Retinal Artery Occlusion, Macular Degeneration (e.g.,age related macular degeneration such as wet AMD or dry AMD),Toxoplasmosis, Choroidal Melanoma, Acquired Retinoschisis, HollenhorstPlaque, Idiopathic Central Serous Chorioretinopathy, Macular Hole,Presumed Ocular Histoplasmosis Syndrome, Retinal Macroaneursym,Retinitis Pigmentosa, Retinal Detachment, Hypertensive Retinopathy,Retinal Pigment Epithelium (RPE) Detachment, Papillophlebitis, OcularIschemic Syndrome, Coats' Disease, Leber's Military Aneurysm,Conjunctival Neoplasms, Allergic Conjunctivitis, Vernal Conjunctivitis,Acute Bacterial Conjunctivitis, Allergic Conjunctivitis &VernalKeratoconjunctivitis, Viral Conjunctivitis, Bacterial Conjunctivitis,Chlamydial & Gonococcal Conjunctivitis, Conjunctival Laceration,Episcleritis, Scleritis, Pingueculitis, Pterygium, Superior LimbicKeratoconjunctivitis (SLK of Theodore), Toxic Conjunctivitis,Conjunctivitis with Pseudomembrane, Giant Papillary Conjunctivitis,Terrien's Marginal Degeneration, Acanthamoeba Keratitis, FungalKeratitis, Filamentary Keratitis, Bacterial Keratitis, KeratitisSicca/Dry Eye Syndrome, Bacterial Keratitis, Herpes Simplex Keratitis,Sterile Corneal Infiltrates, Phlyctenulosis, Corneal Abrasion &Recurrent Corneal Erosion, Corneal Foreign Body, Chemical Burs,Epithelial Basement Membrane Dystrophy (EBMD), Thygeson's SuperficialPunctate Keratopathy, Corneal Laceration, Salzmann's NodularDegeneration, Fuchs' Endothelial Dystrophy, Crystalline LensSubluxation, Ciliary-Block Glaucoma, Primary Open-Angle Glaucoma,Pigment Dispersion Syndrome and Pigmentary Glaucoma, PseudoexfoliationSyndrome and Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary OpenAngle Glaucoma, Uveitic Glaucoma & Glaucomatocyclitic Crisis, PigmentDispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure Glaucoma,Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens InducedGlaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma,Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars Planitis, ChoroidalRupture, Duane's Retraction Syndrome, Toxic/Nutritional OpticNeuropathy, Aberrant Regeneration of Cranial Nerve III, IntracranialMass Lesions, Carotid-Cavernous Sinus Fistula, Anterior Ischemic OpticNeuropathy, Optic Disc Edema & Papilledema, Cranial Nerve III Palsy,Cranial Nerve IV Palsy, Cranial Nerve VI Palsy, Cranial Nerve VII(Facial Nerve) Palsy, Horner's Syndrome, Internuclear Opthalmoplegia,Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve HeadDrusen, Demyelinating Optic Neuropathy (Optic Neuritis, RetrobulbarOptic Neuritis), Amaurosis Fugax and Transient Ischemic Attack,Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal CellCarcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis,Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes SimplexBlepharitis, Orbital Cellulitis, Senile Entropion, and Squamous CellCarcinoma.

By “dermatological disease” as used herein is meant, any disease of thedermis or dermal vasculature, such as skin cancer, rosacea, spiderveins, acne, psoriasis, and excema.

In one embodiment of the present invention, each sequence of an siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs. Exemplary siNA molecules of the invention are shown in TableII. Exemplary synthetic siNA molecules of the invention are shown inTable III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection, with or without their incorporation in biopolymers. Inparticular embodiments, the nucleic acid molecules of the inventioncomprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples ofsuch nucleic acid molecules consist essentially of sequences defined inthese tables and figures. Furthermore, the chemically modifiedconstructs described in Table IV can be applied to any siNA sequence ofthe invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating cancer, proliferative diseases, disorders, orconditions, ocular diseases, disorders, or conditions, dermatologicaldiseases, disorders, or conditions, rheumatoid arthritis, endometriosis,and kidney disease in a subject or organism.

For example, the siNA molecules can be administered to a subject or canbe administered to other appropriate cells evident to those skilled inthe art, individually or in combination with one or more drugs underconditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to prevent or treat cancer, proliferativediseases, disorders, or conditions, ocular diseases, disorders, orconditions, dermatological diseases, disorders, or conditions,rheumatoid arthritis, endometriosis, and kidney disease in a subject ororganism. For example, the described molecules could be used incombination with one or more known compounds, treatments, or proceduresto prevent or treat cancer, proliferative diseases, disorders, orconditions, ocular diseases, disorders, or conditions, dermatologicaldiseases, disorders, or conditions, rheumatoid arthritis, endometriosis,and kidney disease in a subject or organism as are known in the art.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of an siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms an siNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., 2002,Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, NatureBiotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500;and Novina et al., 2002, Nature Medicine, advance online publicationdoi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form ansiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs. Theantisense strand of constructs A-F comprise sequence complementary toany target nucleic acid sequence of the invention. Furthermore, when aglyceryl moiety (L) is present at the 3′-end of the antisense strand forany construct shown in FIG. 4 A-F, the modified internucleotide linkageis optional.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically-modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a PGF-1 siNA sequence. Such chemicalmodifications can be applied to any PGF-1 sequence and/or PGF-1polymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined PGF-1 target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, which is followed by a loop sequence of defined sequence (X),comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in an siNA transcript having specificity for a PGF-1 targetsequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined PGF-1 target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, and which is followed by a 3′-restriction site (R2) which isadjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double-stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palidrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double-strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifunctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifunctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example, a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of NucleicAcid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or an siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of an siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by calorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to an siNA molecule of the invention or thesense and antisense strands of an siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and suitable heterocyclic groups include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl andthe like, all optionally substituted. An “amide” refers to an—C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An“ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylarylor hydrogen.

“Nucleotide” as used herein and as recognized in the art includesnatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to prevent ortreat cancer, proliferative diseases, disorders, or conditions, oculardiseases, disorders, or conditions, dermatological diseases, disorders,or conditions, rheumatoid arthritis, endometriosis, and kidney disease,and/or any other trait, disease, disorder or condition that is relatedto or will respond to the levels of PGF-1 in a cell or tissue, alone orin combination with other therapies. For example, an siNA molecule cancomprise a delivery vehicle, including liposomes, for administration toa subject, carriers and diluents and their salts, and/or can be presentin pharmaceutically acceptable formulations. Methods for the delivery ofnucleic acid molecules are described in Akhtar et al., 1992, Trends CellBio., 2, 139; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol.,16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137,165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all ofwhich are incorporated herein by reference. Beigelman et al, U.S. Pat.No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe thegeneral methods for delivery of nucleic acid molecules. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). Alternatively, thenucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of the invention, whether subcutaneous, intramuscular, orintradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies such as those described inConry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al.,International PCT Publication No. WO 99/31262. The molecules of theinstant invention can be used as pharmaceutical agents. Pharmaceuticalagents prevent, modulate the occurrence, or treat (alleviate a symptomto some extent, preferably all of the symptoms) of a disease state in asubject.

In another embodiment, the nucleic acid molecules of the invention canalso be formulated or complexed with polyethyleneimine and derivativesthereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in United StatesPatent Application Publication No. 20030077829, incorporated byreference herein in its entirety.

In one embodiment, an siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, an siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, the nucleic acid molecules of the invention areadministered via pulmonary delivery, such as by inhalation of an aerosolor spray dried formulation administered by an inhalation device ornebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the nucleicacid compositions of the invention can optionally contain a dispersantwhich serves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition ofthe invention can be produced by any suitable means, such as with anebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers comprise the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride or other suitable salts.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxybenzoate, anti-oxidants,flavorings, volatile oils, buffering agents and emulsifiers and otherformulation surfactants. The aerosols of solid particles comprising theactive composition and surfactant can likewise be produced with anysolid particulate aerosol generator. Aerosol generators foradministering solid particulate therapeutics to a subject produceparticles which are respirable, as explained above, and generate avolume of aerosol containing a predetermined metered dose of atherapeutic composition at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which can be delivered by means of aninsufflator. In the insufflator, the powder, e.g., a metered dosethereof effective to carry out the treatments described herein, iscontained in capsules or cartridges, typically made of gelatin orplastic, which are either pierced or opened in situ and the powderdelivered by air drawn through the device upon inhalation or by means ofa manually-operated pump. The powder employed in the insufflatorconsists either solely of the active ingredient or of a powder blendcomprising the active ingredient, a suitable powder diluent, such aslactose, and an optional surfactant. The active ingredient typicallycomprises from 0.1 to 100 w/w of the formulation. A second type ofillustrative aerosol generator comprises a metered dose inhaler. Metereddose inhalers are pressurized aerosol dispensers, typically containing asuspension or solution formulation of the active ingredient in aliquified propellant. During use these devices discharge the formulationthrough a valve adapted to deliver a metered volume to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, for example US PatentApplication No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728;6,565,885.

In one embodiment, nucleic acid molecules of the invention areadministered to the central nervous system (CNS) or peripheral nervoussystem (PNS). Experiments have demonstrated the efficient in vivo uptakeof nucleic acids by neurons. As an example of local administration ofnucleic acids to nerve cells, Sommer et al., 1998, Antisense Nuc. AcidDrug Dev., 8, 75, describe a study in which a 15mer phosphorothioateantisense nucleic acid molecule to c-fos is administered to rats viamicroinjection into the brain. Antisense molecules labeled withtetramethylrhodamine-isothiocyanate (TRITC) or fluoresceinisothiocyanate (FITC) were taken up by exclusively by neurons thirtyminutes post-injection. A diffuse cytoplasmic staining and nuclearstaining was observed in these cells. As an example of systemicadministration of nucleic acid to nerve cells, Epa et al., 2000,Antisense Nuc. Acid Drug Dev., 10, 469, describe an in vivo mouse studyin which beta-cyclodextrin-adamantane-oligonucleotide conjugates wereused to target the p75 neurotrophin receptor in neuronallydifferentiated PC12 cells. Following a two week course of IPadministration, pronounced uptake of p75 neurotrophin receptor antisensewas observed in dorsal root ganglion (DRG) cells. In addition, a markedand consistent down-regulation of p75 was observed in DRG neurons.Additional approaches to the targeting of nucleic acid to neurons aredescribed in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle etal., 1997, Eur. J. Pharmocol., 340(2/3), 153; Bannai et al., 1998, BrainResearch, 784(1, 2), 304; Rajakumar et al., 1997, Synapse, 26(3), 199;Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998, BrainRes. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience, 74(1), 39.Nucleic acid molecules of the invention are therefore amenable todelivery to and uptake by cells in the CNS and/or PNS.

The delivery of nucleic acid molecules of the invention to the CNS isprovided by a variety of different strategies. Traditional approaches toCNS delivery that can be used include, but are not limited to,intrathecal and intracerebroventricular administration, implantation ofcatheters and pumps, direct injection or perfusion at the site of injuryor lesion, injection into the brain arterial system, or by chemical orosmotic opening of the blood-brain barrier. Other approaches can includethe use of various transport and carrier systems, for example though theuse of conjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

In one embodiment, a compound, molecule, or composition for thetreatment of ocular diseases, disorders and/or conditions (e.g., maculardegeneration, diabetic retinopathy etc.) is administered to a subjectintraocularly or by intraocular means. In another embodiment, acompound, molecule, or composition for the treatment of ocularconditions (e.g., macular degeneration, diabetic retinopathy etc.) isadministered to a subject periocularly or by periocular means (see forexample Ahlheim et al., International PCT publication No. WO 03/24420).In one embodiment, an siNA molecule and/or formulation or compositionthereof is administered to a subject intraocularly or by intraocularmeans. In another embodiment, an siNA molecule and/or formulation orcomposition thereof is administered to a subject periocularly or byperiocular means. Periocular administration generally provides a lessinvasive approach to administering siNA molecules and formulation orcomposition thereof to a subject (see for example Ahlheim et al.,International PCT publication No. WO 03/24420). The use of periocularadministration also minimizes the risk of retinal detachment, allows formore frequent dosing or administration, provides a clinically relevantroute of administration for macular degeneration and other opticconditions, and also provides the possibility of using reservoirs (e.g.,implants, pumps or other devices) for drug delivery.

In one embodiment, delivery systems of the invention include, forexample, aqueous and non-aqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and non-aqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) Cell Fectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NH,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al.; 2001, AAPA Pharm Sci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PharmaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999, PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, an siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. Nos. 6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136;5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as creams, gels, sprays, oilsand other suitable compositions for topical, dermal, or transdermaladministration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexposes the siNA molecules of the invention to an accessible diseasedtissue. The rate of entry of a drug into the circulation has been shownto be a function of molecular weight or size. The use of a liposome orother drug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly(DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, D F et al, 1999, Cell Transplant,8, 47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA, 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly(ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatennary or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pat.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture et al., 1996, TIG.,12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of ansiNA duplex, or a single self-complementary strand that self hybridizesinto an siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi: 10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman-et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of an siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of an siNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region in a manner whichallows expression and/or delivery of the siNA molecule.

PGF-1 Biology and Biochemistry

Angiogenesis is a process of new blood vessel development frompre-existing vasculature. It plays an essential role in embryonicdevelopment, normal growth of tissues, wound healing, the femalereproductive cycle (i.e., ovulation, menstruation and placentaldevelopment), as well as a major role in many diseases. Particularinterest has focused on cancer, since tumors cannot grow beyond a fewmillimeters in size without developing a new blood supply. Angiogenesisis also necessary for the spread and growth of tumor cell metastases.

One important growth and survival factor for tumor progression isplacental growth factor (PGF-1). Placental growth factor (PGF, alsoknown as PlGF) is a homolog of VEGF, that stimulates revascularizationof ischemic tissues. There are three isoforms of PGF (PGF-1, PGF-2, andPGF-3). Through dimerization upon ligand binding, VEGF and PGF bindtransmember receptor tyrosine kinases that juxtapose cytoplasmictyrosine kinase domains which in turn transphosphorylate tyrosineresidues in a partner molecule (intramolecular cross talk). PGFregulates the cross talk between VEGF receptors VEGFR-1 and VEGFR-2.VEGFR-1 transmits signals for the activation of angiogenesis. Theactivation of VEGFR-1 by PGF results in the intermoleculartransphosphorylation of VEGFR-2, which is able to amplify VEGF drivenangiogenesis through VEGFR-2 transduction. VEGF and PGF both bindVEGFR-1; however, PGF is able to uniquely stimulate the phosphorylationof specific VEGFR-1 tyrosine residues and the expression of distinctdownstream target genes (Autiero et. al., 2003, Nature Medicine 9:936-943).

PGF can trigger its own intracellular signals independent ofVEGF/VEGFR-2 signaling. Even though PGF-2 and VEGF each stimulatedVEGFR-1 tyrosine phosphorylation, only PGF rescues the impaired VEGFsurvival response of PGF capillary endothelial cells (CEC's) throughincreased activation of VEGFR-1/VEGFR-2 heterodimers. In addition,VEGF/PGF heterodimers can stimulate angiogenesis through the formationof VEGFR-1/VEGFR-2 heterodimers. PGF can also enhance VEGFR-1transphosphorylation through activation of VEGFR-1, while VEGFsuppresses VEGFR-1 transphosphorylation. As such, PGF is able to amplifyVEGF driven angiogenesis through inter- and intramolecular cross talkbetween VEGFR-1 and VEGFR-2 (Autiero et. al., 2003, Nature Medicine 9:936-943).

Agents that interfere with blood vessel formation can be used to blocktumor angiogenesis, tumor progression and metastasis. The use of smallinterfering nucleic acid molecules targeting PGF-1, therefore provides aclass of novel therapeutic agents that can be used in the treatment,alleviation, or prevention of cancer, proliferative diseases, disorders,or conditions, ocular diseases, disorders, or conditions, dermatologicaldiseases, disorders, or conditions, rheumatoid arthritis, endometriosis,and kidney disease, alone or in combination with other therapies.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5 M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H2O followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables II and III). Ifterminal TT residues are desired for the sequence (as described inparagraph 7), then the two 3′ terminal nucleotides of both the sense andantisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see, for example, Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a PGF-1target sequence is used to screen for target sites in cells expressingPGF-1 RNA, such as such HUVEC, HMVEC, A549, HELA, A375, or capillaryendothelial cells. The general strategy used in this approach is shownin FIG. 9. A non-limiting example of such is a pool comprising sequenceshaving any of SEQ ID NOS 1-316. Cells expressing PGF-1 are transfectedwith the pool of siNA constructs and cells that demonstrate a phenotypeassociated with PGF-1 inhibition are sorted. The pool of siNA constructscan be expressed from transcription cassettes inserted into appropriatevectors (see for example FIG. 7 and FIG. 8). The siNA from cellsdemonstrating a positive phenotypic change (e.g., decreasedproliferation, decreased PGF-1 mRNA levels or decreased PGF-1 proteinexpression), are sequenced to determine the most suitable target site(s)within the target PGF-1 RNA sequence.

Example 4 PGF-1 Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the PGF-1 RNAtarget and optionally prioritizing the target sites on the basis offolding (structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. Nos. 5,831,071,6,353,098, 6,437,117, and Bellon et al., U.S. Pat. Nos. 6,054,576,6,162,909, 6,303,773, or Scaringe supra, incorporated by referenceherein in their entireties. Additionally, deprotection conditions can bemodified to provide the best possible yield and purity of siNAconstructs. For example, applicant has observed that oligonucleotidescomprising 2′-deoxy-2′-fluoro nucleotides can degrade underinappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi in Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting PGF-1 RNA targets. The assaycomprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with PGF-1 target RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriatePGF-1 expressing plasmid using T7 RNA polymerase or via chemicalsynthesis as described herein. Sense and antisense siNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing siNA (10 nM final concentration). Thereaction mixture also contains 10 mM creatine phosphate, 10 ug/mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25× Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in thePGF-1 RNA target for siNA mediated RNAi cleavage, wherein a plurality ofsiNA constructs are screened for RNAi mediated cleavage of the PGF-1 RNAtarget, for example, by analyzing the assay reaction by electrophoresisof labeled target RNA, or by northern blotting, as well as by othermethodology well known in the art.

Example 7 Nucleic Acid Inhibition of PGF-1 Target RNA

siNA molecules targeted to the human PGF-1 RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within thePGF-1 RNA are given in Tables II and III.

Two formats are used to test the efficacy of siNAs targeting PGF-1.First, the reagents are tested in cell culture using, for example,HUVEC, HMVEC, A549, HELA, A375, or capillary endothelial cells, todetermine the extent of RNA and protein inhibition. siNA reagents (e.g.;see Tables II and III) are selected against the PGF-1 target asdescribed herein. RNA inhibition is measured after delivery of thesereagents by a suitable transfection agent to, for example, HUVEC, HMVEC,A549, HELA, A375, or capillary endothelial cells. Relative amounts oftarget RNA are measured versus actin using real-time PCR monitoring ofamplification (e.g., ABI 7700 TAQMAN®). A comparison is made to amixture of oligonucleotide sequences made to unrelated targets or to arandomized siNA control with the same overall length and chemistry, butrandomly substituted at each position. Primary and secondary leadreagents are chosen for the target and optimization performed. After anoptimal transfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead siNA molecule. In addition, acell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells such as HUVEC, HMVEC, A549, HELA, A375, or capillary endothelialcells are seeded, for example, at 1×10⁵ cells per well of a six-welldish in EGM-2 (BioWhittaker) the day before transfection. siNA (finalconcentration, for example 20 nM) and cationic lipid (e.g., finalconcentration 2 μg/nm) are complexed in EGM basal media (Bio Whittaker)at 37° C. for 30 minutes in polystyrene tubes. Following vortexing, thecomplexed siNA is added to each well and incubated for the timesindicated. For initial optimization experiments, cells are seeded, forexample, at 1×10³ in 96 well plates and siNA complex added as described.Efficiency of delivery of siNA to cells is determined using afluorescent siNA complexed with lipid. Cells in 6-well dishes areincubated with siNA for 24 hours, rinsed with PBS and fixed in 2%paraformaldehyde for 15 minutes at room temperature. Uptake of siNA isvisualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, IOU RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted-total cellular RNA(300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitrocellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Animal Models Useful to Evaluate the Down-Regulation of PGF-1Gene Expression

There are several animal models in which the anti-angiogenesis effect ofnucleic acids of the present invention, such as siNA, directed againstPGF-1 mRNAs can be tested. Typically a corneal model has been used tostudy angiogenesis in rat and rabbit since recruitment of vessels caneasily be followed in this normally avascular tissue (Pandey et al.,1995 Science 268: 567-569). In these models, a small Teflon or Hydrondisk pretreated with an angiogenesis factor (e.g. bFGF or VEGF) isinserted into a pocket surgically created in the cornea. Such models canbe adapted for use with siNA molecules targeting PGF-1, for example byusing a Teflon or Hydron disk pretreated with PGF-1, or by using theVEGF treated disk for studying the effects of PGF-1 inhibition on VEGFinduced angiogenesis. Using these models, angiogenesis is typicallymonitored 3 to 5 days after implantation. In a non-limiting example,siNA directed against PGF-1 mRNAs are delivered in the disk as well, ordropwise to the eye over the time course of the experiment. In anothereye model, hypoxia has been shown to cause both increased expression ofVEGF and neovascularization in the retina (Pierce et al., 1995 Proc.Natl. Acad. Sci. USA. 92: 905-909; Shweiki et al., 1992 J. Clin. Invest.91: 2235-2243). Similarly, this model can be used to study the effectsof PGF-1 inhibition on hypoxia induced angiogenesis.

In human glioblastomas, it has been shown that VEGF is at leastpartially responsible for tumor angiogenesis (Plate et al., 1992 Nature359, 845). Animal models have been developed in which glioblastoma cellsare implanted subcutaneously into nude mice and the progress of tumorgrowth and angiogenesis is studied (Kim et al., 1993 supra; Millauer etal., 1994 supra). This model can be used to study the effects of PGF-1inhibition on tumor angiogenesis.

Another animal model that addresses neovascularization involvesMatrigel, an extract of basement membrane that becomes a solid gel wheninjected subcutaneously (Passaniti et al., 1992 Lab. Invest. 67:519-528). When the Matrigel is supplemented with angiogenesis factorssuch as VEGF, vessels grow into the Matrigel over a period of 3 to 5days and angiogenesis can be assessed. Again, nucleic acids directedagainst PGF-1 mRNAs are delivered in the Matrigel and this model can beused to study the effects of PGF-1 inhibition on tumor angiogenesis,metastasis, and neovascularization.

Several animal models exist for screening of anti-angiogenic agents.These include corneal vessel formation following corneal injury (Burgeret al., 1985 Cornea 4: 35-41; Lepri, et al., 1994 J. Ocular Pharmacol.10: 273-280; Ormerod et al., 1990 Am. J. Pathol 137: 1243-1252) orintracorneal growth factor implant (Grant et al., 1993 Diabetologia 36:282-291; Pandey et al. 1995 supra; Zieche et al., 1992 Lab. Invest. 67:711-715), vessel growth into Matrigel matrix containing growth factors(Passaniti et al., 1992 supra), female reproductive organneovascularization following hormonal manipulation (Shweiki et al., 1993Clin. Invest. 91: 2235-2243), several models involving inhibition oftumor growth in highly vascularized solid tumors (O'Reilly et al., 1994Cell 79: 315-328; Senger et al., 1993 Cancer and Metas. Rev. 12:303-324; Takahasi et al., 1994 Cancer Res. 54: 4233-4237; Kim et al.,1993 supra), and transient hypoxia-induced neovascularization in themouse retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92:905-909). Other model systems to study tumor angiogenesis are reviewedby Folkman, 1985 Adv. Cancer. Res. 43, 175. All of these models can beused to study the efficacy of siNA molecules of the invention intreating angiogenesis related disease.

Ocular Models of Angiogenesis

The cornea model, described in Pandey et al. supra, is the most commonand well characterized model for screening anti-angiogenic agentefficacy. This model involves an avascular tissue into which vessels arerecruited by a stimulating agent (growth factor, thermal or alkali burn,endotoxin). The corneal model utilizes the intrastromal cornealimplantation of a Teflon pellet soaked in a VEGF-Hydron solution torecruit blood vessels toward the pellet, which can be quantitated usingstandard microscopic and image analysis techniques. To evaluate theiranti-angiogenic efficacy, nucleic acids are applied topically to the eyeor bound within Hydron on the Teflon pellet itself. This avascularcornea as well as the Matrigel (see below) provide for low backgroundassays. While the corneal model has been performed extensively in therabbit, studies in the rat have also been conducted. This model can bereadily adapted for use with siNA molecules of the invention targetingPGF-1, such as in examining the effect of PGF-1 inhibition on VEGFinduced angiogenesis, or by using an PGF-1 pellet to specificallydetermine the effect of PGF-1 inhibition on PGF-1 induced angiogenesis.

The mouse model (Passaniti et al., supra) is a non-tissue model thatutilizes Matrigel, an extract of basement membrane (Kleinman et al.,1986) or Millipore® filter disk, which can be impregnated with growthfactors and anti-angiogenic agents in a liquid form prior to injection.Upon subcutaneous administration at body temperature, the Matrigel orMillipore® filter disk forms a solid implant. VEGF embedded in theMatrigel or Millipore® filter disk is used to recruit vessels within thematrix of the Matrigel or Millipore® filter disk which can be processedhistologically for endothelial cell specific vWF (factor VIII antigen)immunohistochemistry, Trichrome-Masson stain, or hemoglobin content.Like the cornea, the Matrigel or Millipore® filter disk is avascular;however, it is not tissue. In the Matrigel or Millipore® filter diskmodel, nucleic acids are administered within the matrix of the Matrigelor Millipore® filter disk to test their anti-angiogenic efficacy. Thus,delivery issues in this model, as with delivery of nucleic acids byHydron-coated Teflon pellets in the rat cornea model, may be lessproblematic due to the homogeneous presence of the nucleic acid withinthe respective matrix. Similarly, this model can be readily adapted foruse with siNA molecules of the invention targeting PGF-1, such as inexamining the effect of PGF-1 inhibition on VEGF induced angiogenesis,or by using an PGF-1 filter disk to specifically determine the effect ofPGF-1 inhibition on PGF-1 induced angiogenesis.

Additionally, siNA molecules of the invention targeting PGF-1 can beassessed for activity using transgenic mice to determine whethermodulation of PGF-1 can inhibit optic neovascularization. Animal modelsof choroidal neovascularization are described in, for example, Mori etal., 2001, Journal of Cellular Physiology, 188, 253; Mori et al., 2001,American Journal of Pathology, 159, 313; Ohno-Matsui et al., 2002,American Journal of Pathology, 160, 711; and Kwak et al., 2000,Investigative Opthalmology & Visual Science, 41, 3158. These models canbe used to assess the efficacy of PGF-1 inhibition on opticneovascularization.

In a non-limiting example, CNV is laser induced in, for example, adultC57BL/6 mice. The mice are also given an intravitreous, periocular or asubretinal injection of PGF-1 siNA in each eye. Intravitreous injectionsare made using a Harvard pump microinjection apparatus and pulled glassmicropipets. Then a micropipette is passed through the sclera justbehind the limbus into the vitreous cavity. The subretinal injectionsare made using a condensing lens system on a dissecting microscope. Thepipet tip is then passed through the sclera posterior to the limbus andpositioned above the retina. Five days after the injection of the vectorthe mice are anesthetized with ketamine hydrochloride (100 mg/kg bodyweight), 1% tropicamide is also used to dilate the pupil, and a diodelaser photocoagulation is used to rupture Bruch's membrane at threelocations in each eye. A slit lamp delivery system and a hand-held coverslide are used for laser photocoagulation. Burns are made in the 9, 12,and 3 o'clock positions 2-3 disc diameters from the optic nerve (Mori etal., supra).

The mice typically develop subretinal neovascularization due to theexpression of VEGF in photoreceptors beginning at prenatal day 7. Atprenatal day 21, the mice are anesthetized and perfused with 1 ml ofphosphate-buffered saline containing 50 mg/ml of fluorescein-labeleddextran. Then the eyes are removed and placed for 1 hour in a 10%phosphate-buffered formalin. The retinas are removed and examined byfluorescence microscopy (Mori et al., supra).

Fourteen days after the laser induced rupture of Bruch's membrane, theeyes that received intravitreous and subretinal injection of siNA areevaluated for smaller appearing areas of CNV, while control eyes areevaluated for large areas of CNV. The eyes that receive intravitreousinjections or a subretinal injection of siNA are also evaluated forfewer areas of neovascularization on the outer surface of the retina andpotential abortive sprouts from deep retinal capillaries that do notreach the retinal surface compared to eyes that did not receive aninjection of siNA.

Tumor Models of Angiogenesis

Use of Murine Models

For a typical systemic study involving 10 mice (20 g each) per dosegroup, 5 doses (1, 3, 10, 30 and 100 mg/kg daily over 14 days continuousadministration), approximately 400 mg of siNA, formulated in saline isused. A similar study in young adult rats (200 g) can require over 4 gof siNA. Parallel pharmacokinetic studies involve the use of similarquantities of siNA further justifying the use of murine models.

Lewis Lung Carcinoma and B-16 Melanoma Murine Models

Identifying a common animal model for systemic efficacy testing ofnucleic acids is an efficient way of screening siNA for systemicefficacy.

The Lewis lung carcinoma and B-16 murine melanoma models are wellaccepted models of primary and metastatic cancer and are used forinitial screening of anti-cancer agents. These murine models are notdependent upon the use of immunodeficient mice, are relativelyinexpensive, and minimize housing concerns. Both the Lewis lung and B-16melanoma models involve subcutaneous implantation of approximately 10⁶tumor cells from metastatically aggressive tumor cell lines (Lewis lunglines 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice.Alternatively, the Lewis lung model can be produced by the surgicalimplantation of tumor spheres (approximately 0.8 mm in diameter).Metastasis also can be modeled by injecting the tumor cells directlyintravenously. In the Lewis lung model, microscopic metastases can beobserved approximately 14 days following implantation with quantifiablemacroscopic metastatic tumors developing within 21-25 days. The B-16melanoma exhibits a similar time course with tumor neovascularizationbeginning 4 days following implantation. Since both primary andmetastatic tumors exist in these models after 21-25 days in the sameanimal, multiple measurements can be taken as indices of efficacy.Primary tumor volume and growth latency as well as the number of micro-and macroscopic metastatic lung foci or number of animals exhibitingmetastases can be quantitated. The percent increase in lifespan can alsobe measured. Thus, these models provide suitable primary efficacy assaysfor screening systemically administered siNA nucleic acids and siNAnucleic acid formulations.

In the Lewis lung and B-16 melanoma models, systemic pharmacotherapywith a wide variety of agents usually begins 1-7 days following tumorimplantation/inoculation with either continuous or multipleadministration regimens. Concurrent pharmacokinetic studies can beperformed to determine whether sufficient tissue levels of siNA can beachieved for pharmacodynamic effect to be expected. Furthermore, primarytumors and secondary lung metastases can be removed and subjected to avariety of in vitro studies (i.e. target RNA reduction).

In addition, animal models are useful in screening compounds, e.g., siNAmolecules, for efficacy in treating renal failure, such as a result ofautosomal dominant polycystic kidney disease (ADPKD). The Han:SPRD ratmodel, mice with a targeted mutation in the Pkd2 gene and congenitalpolycystic kidney (cpk) mice, closely resemble human ADPKD and provideanimal models to evaluate the therapeutic effect of siRNA constructsthat have the potential to interfere with one or more of the pathogenicelements of ADPKD mediated renal failure, such as angiogenesis.Angiogenesis may be necessary in the progression of ADPKD for growth ofcyst cells as well as increased vascular permeability promoting fluidsecretion into cysts. The use of Han:SPRD rats (see for exampleKaspareit-Rittinghausen et al., 1991, Am. J. Pathol. 139, 693-696), micewith a targeted mutation in the Pkd2 gene (Pkd2−/− mice, see for exampleWu et al., 2000, Nat. Genet. 24, 75-78) and cpk mice (see for exampleWoo et al., 1994, Nature, 368, 750-753) all provide animal models thatcan be used to study the efficacy of siNA molecules of the inventionagainst PGF-1 mediated renal failure.

PGF-1 protein levels can be measured clinically or experimentally byFACS analysis. PGF-1 encoded mRNA levels are assessed by Northernanalysis, RNase-protection, primer extension analysis and/orquantitative RT-PCR. siRNA nucleic acids that block PGF-1 proteinencoding mRNAs and therefore result in decreased levels of PGF-1activity by more than 20% in vitro can be identified.

Restenosis

Smooth muscle cell (SMC) proliferation is an important component ofrestenosis in response to injury after balloon angioplasty. The ratcarotid artery balloon injury model is a well-characterized and highlyreproducible vascular proliferative disorder that is dependent on SMCmigration and proliferation. Accordingly, this model can be used todetermine whether the inhibition of SMC proliferation by siNA observedin cell culture experiments is sufficient to impact neointima formationin a rat carotid artery model of balloon angioplasty. Rat carotidarteries are subjected to balloon angioplasty and immediately exposed tosiNA or inactive control siNA molecules. In a non-limiting example,Sprague-Dawley rats are subjected to balloon angioplasty of the leftcommon carotid artery by dilatation with a Fogarty catheter. Immediatelyfollowing injury, siNA, or inactive control siNA molecules in a volumeof 50 to 100 μl is instilled into a 1-cm segment of the distal commoncarotid artery for 5 minutes by using a 24-gauge intravenous catheter.Rat carotid arteries are harvested 20 days after balloon injury andadenovirus infection. Tissue sections are stained with hematoxylin andeosin. Intimal and medial boundaries are determined by digitalplanimetry of tissue sections. Areas and ratios are determined from fourto six stained sections of each artery spanning the 1-cm site of ballooninjury. As such, the efficacy of siNA molecules of the invention can bedetermined via inhibition of smooth muscle cell proliferation in the ratcarotid artery balloon injury model.

Example 9 RNAi Mediated Inhibition of PGF-1 Expression

siNA constructs (Table III) are tested for efficacy in reducing PGF-1RNA expression in, for example, HUVEC, HMVEC, A549, HELA, A375, orcapillary endothelial cells. Cells are plated approximately 24 hoursbefore transfection in 96-well plates at 5,000-7,500 cells/well, 100μl/well, such that at the time of transfection cells are 70-90%confluent. For transfection, annealed siNAs are mixed with thetransfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50μl/well and incubated for 20 minutes at room temperature. The siNAtransfection mixtures are added to cells to give a final siNAconcentration of 25 nM in a volume of 150 μl. Each siNA transfectionmixture is added to 3 wells for triplicate siNA treatments. Cells areincubated at 37° for 24 hours in the continued presence of the siNAtransfection mixture. At 24 hours, RNA is prepared from each well oftreated cells. The supernatants with the transfection mixtures are firstremoved and discarded, then the cells are lysed and RNA prepared fromeach well. Target gene expression following treatment is evaluated byRT-PCR for the target gene and for a control gene (36B4, an RNApolymerase subunit) for normalization. The triplicate data is averagedand the standard deviations determined for each treatment. Normalizeddata are graphed and the percent reduction of target mRNA by activesiNAs in comparison to their respective inverted control siNAs isdetermined.

Example 10 Indications

The present body of knowledge in PGF-1 research indicates the need formethods to assay PGF-1 activity and for compounds that can regulatePGF-1 expression for research, diagnostic, and therapeutic use. Asdescribed herein, the nucleic acid molecules of the present inventioncan be used in assays to diagnose disease state related of PGF-1 levels.In addition, the nucleic acid molecules can be used to treat diseasestate related to PGF-1 levels.

Particular conditions and disease states that can be associated withPGF-1 expression modulation include, but are not limited to:

1) Tumor angiogenesis: Angiogenesis has been shown to be necessary fortumors to grow into pathological size (Folkman, 1971, PNAS 76,5217-5221; Wellstein & Czubayko, 1996, Breast Cancer Res and Treatment38, 109-119). In addition, it allows tumor cells to travel through thecirculatory system during metastasis. Increased levels of geneexpression of a number of angiogenic factors such as placental growthfactor (PGF-1) have been reported in vascularized and edema-associatedbrain tumors (Berkman et al., 1993 J. Clini. Invest. 91, 153). A moredirect demonstration of the role of PGF-1 in tumor angiogenesis wasdemonstrated by Jim Kim et al., 1993 Nature 362,841 wherein, monoclonalantibodies against PGF-1 were successfully used to inhibit the growth ofrhabdomyosarcoma, glioblastoma multiform cells in nude mice. Similarly,expression of a dominant negative mutated form of the flt-1 PGF-1receptor inhibits vascularization induced by human glioblastoma cells innude mice (Millauer et al., 1994, Nature 367, 576). Specifictumor/cancer types that can be targeted using the nucleic acid moleculesof the invention include but are not limited to the tumor/cancer typesdescribed herein.

2) Ocular diseases: Neovascularization has been shown to cause orexacerbate ocular diseases including, but not limited to, maculardegeneration, neovascular glaucoma, diabetic retinopathy, myopicdegeneration, and trachoma (Norrby, 1997, APMIS 105, 417-437). Aiello etal., 1994 New Engl. J. Med. 331, 1480, showed that the ocular fluid of amajority of patients suffering from diabetic retinopathy and otherretinal disorders contains a high concentration of PGF-1. Miller et al.,1994 Am. J. Pathol. 145, 574, reported elevated levels of PGF-1 mRNA inpatients suffering from retinal ischemia. These observations support adirect role for PGF-1 in ocular diseases. Other factors, including thosethat stimulate PGF-1 synthesis, may also contribute to theseindications.

3) Dermatological Disorders: Many indications have been identified whichmay be angiogenesis dependent, including but not limited to, psoriasis,verruca vulgaris, angiofibroma of tuberous sclerosis, pot-wine stains,Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome, andOsler-Weber-Rendu syndrome (Norrby, supra). Intradermal injection of theangiogenic factor b-FGF demonstrated angiogenesis in nude mice(Weckbecker et al., 1992, Angiogenesis: Keyprinciples-Science-Technology-Medicine, ed R. Steiner). Detmar et al.,1994 J. Exp. Med. 180, 1141 reported that PGF-1 and its receptors wereover-expressed in psoriatic skin and psoriatic dermal microvessels,suggesting that PGF-1 plays a significant role in psoriasis.

4) Rheumatoid arthritis: Immunohistochemistry and in situ hybridizationstudies on tissues from the joints of patients suffering from rheumatoidarthritis show an increased level of PGF-1 and its receptors (Fava etal., 1994 J. Exp. Med. 180, 341). Additionally, Koch et al., 1994 J.Immunol. 152, 4149, found that PGF-1-specific antibodies were able tosignificantly reduce the mitogenic activity of synovial tissues frompatients suffering from rheumatoid arthritis. These observations supporta direct role for PGF-1 in rheumatoid arthritis. Other angiogenicfactors including those of the present invention may also be involved inarthritis.

5) Endometriosis: Various studies indicate that PGF-1 is directlyimplicated in endometriosis. In one study, PGF-1 concentrations measuredby ELISA in peritoneal fluid were found to be significantly higher inwomen with endometriosis than in women without endometriosis (24.1±15ng/ml vs 13.3±7.2 ng/ml in normals). In patients with endometriosis,higher concentrations of PGF-1 were detected in the proliferative phaseof the menstrual cycle (33±13 ng/ml) compared to the secretory phase(10.7±5 ng/ml). The cyclic variation was not noted in fluid from normalpatients (McLaren et al., 1996, Human Reprod. 11, 220-223). In anotherstudy, women with moderate to severe endometriosis had significantlyhigher concentrations of peritoneal fluid PGF-1 than women withoutendometriosis. There was a positive correlation between the severity ofendometriosis and the concentration of PGF-1 in peritoneal fluid. Inhuman endometrial biopsies, PGF-1 expression increased relative to theearly proliferative phase approximately 1.6-, 2-, and 3.6-fold inmidproliferative, late proliferative, and secretory endometrium (Shifrenet al., 1996, J. Clin. Endocrinol. Metab. 81, 3112-3118). In a thirdstudy, PGF-1-positive staining of human ectopic endometrium was shown tobe localized to macrophages (double immunofluorescent staining with CD14marker). Peritoneal fluid macrophages demonstrated PGF-1 staining inwomen with and without endometriosis. However, increased activation ofmacrophages (acid phosphatase activity) was demonstrated in fluid fromwomen with endometriosis compared with controls. Peritoneal fluidmacrophage conditioned media from patients with endometriosis resultedin significantly increased cell proliferation ([³H] thymidineincorporation) in HUVEC cells compared to controls. The percentage ofperitoneal fluid macrophages with PGF-1 mRNA was higher during thesecretory phase, and significantly higher in fluid from women withendometriosis (80±15%) compared with controls (32±20%). Flt-mRNA wasdetected in peritoneal fluid macrophages from women with and withoutendometriosis, but there was no difference between the groups or anyevidence of cyclic dependence (McLaren et al., 1996, J. Clin. Invest.98, 482-489). In the early proliferative phase of the menstrual cycle,PGF-1 has been found to be expressed in secretory columnar epithelium(estrogen-responsive) lining both the oviducts and the uterus in femalemice. During the secretory phase, PGF-1 expression was shown to haveshifted to the underlying stroma composing the functional endometrium.In addition to examining the endometrium, neovascularization of ovarianfollicles and the corpus luteum, as well as angiogenesis in embryonicimplantation sites have been analyzed. For these processes, PGF-1 wasexpressed in spatial and temporal proximity to forming vasculature(Shweiki et al., 1993, J. Clin. Invest. 91, 2235-2243).

6) Kidney disease: Autosomal dominant polycystic kidney disease (ADPKD)is the most common life threatening hereditary disease in the USA. Itaffects about 1:400 to 1:1000 people and approximately 50% of peoplewith ADPKD develop renal failure. ADPKD accounts for about 5-10% ofend-stage renal failure in the USA, requiring dialysis and renaltransplantation. Angiogenesis is implicated in the progression of ADPKDfor growth of cyst cells, as well as increased vascular permeabilitypromoting fluid secretion into cysts. Proliferation of cystic epitheliumis a feature of ADPKD because cyst cells in culture produce solubleplacental growth factor (PGF-1). PGF-1 has been detected in epithelialcells of cystic tubules but not in endothelial cells in the vasculatureof cystic kidneys or normal kidneys. PGF-1 expression is increased inendothelial cells of cyst vessels and in endothelial cells during renalischemia-reperfusion.

The use of radiation treatments and chemotherapeutics, such asGemcytabine and cyclophosphamide, are non-limiting examples ofchemotherapeutic agents that can be combined with or used in conjunctionwith the nucleic acid molecules (e.g. siNA molecules) of the instantinvention. Those skilled in the art will recognize that otheranti-cancer compounds and therapies can similarly be readily combinedwith the nucleic acid molecules of the instant invention (e.g. siNAmolecules) and are hence within the scope of the instant invention. Suchcompounds and therapies are well known in the art (see for exampleCancer: Principles and Practice of Oncology, Volumes 1 and 2, edsDevita, V. T., Hellman, S., and Rosenberg, S. A., J.B. LippincottCompany, Philadelphia, USA; incorporated herein by reference) andinclude, without limitation, folates, antifolates, pyrimidine analogs,fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase Iinhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins,platinum analogs, alkylating agents, nitrosoureas, plant derivedcompounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinaseinhibitors, taxols, radiation therapy, surgery, nutritional supplements,gene therapy, radiotherapy, for example 3D-CRT, immunotoxin therapy, forexample ricin, and monoclonal antibodies. Specific examples ofchemotherapeutic compounds that can be combined with or used inconjunction with the nucleic acid molecules of the invention include,but are not limited to, Paclitaxel; Docetaxel; Methotrexate; Doxorubin;Edatrexate; Vinorelbine; Tamoxifen; Leucovorin; 5-fluoro uridine (5-FU);Ionotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin;Mitomycin C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine;L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan;Ifosfamide; 4-hydroperoxycyclophosphamide; Thiotepa; Irinotecan(CAMPTOSAR®, CPT-11, Camptothecin-11, Campto) Tamoxifen; Herceptin; IMCC225; ABX-EGF; and combinations thereof. The above list of compounds arenon-limiting examples of compounds and/or methods that can be combinedwith or used in conjunction with the nucleic acid molecules (e.g. siNA)of the instant invention. Those skilled in the art will recognize thatother drug compounds and therapies can similarly be readily combinedwith the nucleic acid molecules of the instant invention (e.g., siNAmolecules) are hence within the scope of the instant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with an siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I PGF Accession Numbers NM_002632 Homo sapiens placental growthfactor, vascular endothelial growth factor-related protein (PGF), mRNAgi|34147600|ref|NM_002632.3|[34147600] BC001422 Homo sapiens placentalgrowth factor, vascular endothelial growth factor-related protein, mRNA(cDNA clone MGC: 1683 IMAGE: 3139175), complete cdsgi|33876199|gb|BC001422.2|[33876199] BC007255 Homo sapiens, clone MGC:15513 IMAGE: 3028008, mRNA, complete cdsgi|13938258|gb|BC007255.1|BC007255[13938258] BC007789 Homo sapiensplacental growth factor, vascular endothelial growth factor-relatedprotein, mRNA (cDNA clone MGC: 14095 IMAGE: 3629125), complete cdsgi|33987939|gb|BC007789.2|[33987939] AC006530 Homo sapiens chromosome 14clone BAC 316E14 map 14q24.3, complete sequencegi|4680764|gb|AC006530.4|AC006530[4680764] X54936 H. sapiens mRNA forplacenta growth factor (PlGF) gi|35521|emb|X54936.1|HSPLGF[35521]BX248289 human full-length cDNA clone CS0DI075YC18 of Placenta of Homosapiens (human) gi|28207926|emb|BX248289.1|CNSLT1I9B[28207926] S72960Homo sapiens placenta growth factor 2 (PlGF-2) mRNA, complete cdsgi|633813|bbm|348514|bbs|154805|gb|S72960.1|S72960[633813] BT007182 Homosapiens placental growth factor, vascular endothelial growthfactor-related protein mRNA, complete cdsgi|30583202|gb|BT007182.1|[30583202] BT008273 Synthetic construct Homosapiens placental growth factor, vascular endothelial growthfactor-related protein mRNA, partial cdsgi|30585384|gb|BT008273.1|[30585384] BC007255 Homo sapiens, clone MGC:15513 IMAGE: 3028008, mRNA, complete cdsgi|13938258|gb|BC007255.1|BC007255[13938258]

TABLE II PGF siNA AND TARGET SEQUENCES PGF NM_002632 Seq Seq Seq Pos SeqID UPos Upper seq ID LPos Lower seq ID    3 GCUGUCUGCGGAGGAAACU 1 3GCUGUCUGCGGAGGAAACU 1 21 AGUUUCCUCCGCAGACAGC 98   21 UGCAUCGACGGACGGCCGC2 21 UGCAUCGACGGACGGCCGC 2 39 GCGGCCGUCCGUCGAUGCA 99   39CCCAGCUACGGGAGGACCU 3 39 CCCAGCUACGGGAGGACCU 3 57 AGGUCCUCCCGUAGCUGGG100   57 UGGAGUGGCACUGGGCGCC 4 57 UGGAGUGGCACUGGGCGCC 4 75GGCGCCCAGUGCCACUCCA 101   75 CCGACGGACCAUCCCCGGG 5 75CCGACGGACCAUCCCCGGG 5 93 CCCGGGGAUGGUCCGUCGG 102   93GACCCGCCUGCCCCUCGGC 6 93 GACCCGCCUGCCCCUCGGC 6 111 GCCGAGGGGCAGGCGGGUC103  111 CGCCCCGCCCCGCCGGGCC 7 111 CGCCCCGCCCCGCCGGGCC 7 129GGCCGGGCGGGGCGGGGCG 104  129 CGCUCCCCGUCGGGUUCCC 8 129CGCUCCCCGUCGGGUUCCC 8 147 GGGAACCCGACGGGGAGCG 105  147CCAGCCACAGCCUUACCUA 9 147 CCAGCCACAGCCUUACCUA 9 165 UAGGUAAGGCUGUGGCUGG106  165 ACGGGCUCCUGACUCCGCA 10 165 ACGGGCUCCUGACUCCGCA 10 183UGCGGAGUCAGGAGCCCGU 107  183 AAGGCUUCCAGAAGAUGCU 11 183AAGGCUUCCAGAAGAUGCU 11 201 AGCAUCUUCUGGAAGCCUU 108  201UCGAACCACCGGCCGGGGC 12 201 UCGAACCACCGGCCGGGGC 12 219GCCCCGGCCGGUGGUUCGA 109  219 CCUCGGGGCAGCAGUGAGG 13 219CCUCGGGGCAGCAGUGAGG 13 237 CCUCACUGCUGCCCCGAGG 110  237GGAGGCGUCCAGCCCCCCA 14 237 GGAGGCGUCCAGCCCCCCA 14 255UGGGGGGCUGGACGCCUCC 111  255 ACUCAGCUCUUCUCCUCCU 15 255ACUCAGCUCUUCUCCUCCU 15 273 AGGAGGAGAAGAGCUGAGU 112  273UGUGCCAGGGGCUCCCCGG 16 273 UGUGCCAGGGGCUCCCCGG 16 291CCGGGGAGCCCCUGGCACA 113  291 GGGGAUGAGCAUGGUGGUU 17 291GGGGAUGAGCAUGGUGGUU 17 309 AACCACCAUGCUCAUCCCC 114  309UUUCCCUCGGAGCCCCCUG 18 309 UUUCCCUCGGAGCCCCCUG 18 327CAGGGGGCUCCGAGGGAAA 115  327 GGCUCGGGACGUCUGAGAA 19 327GGCUCGGGACGUCUGAGAA 19 345 UUCUCAGACGUCCCGAGCC 116  345AGAUGCCGGUCAUGAGGCU 20 345 AGAUGCCGGUCAUGAGGCU 20 363AGCCUCAUGACCGGCAUCU 117  363 UGUUCCCUUGCUUCCUGCA 21 363UGUUCCCUUGCUUCCUGCA 21 381 UGCAGGAAGCAAGGGAACA 118  381AGCUCCUGGCCGGGCUGGC 22 381 AGCUCCUGGCCGGGCUGGC 22 399GCCAGCCCGGCCAGGAGCU 119  399 CGCUGCCUGCUGUGCCCCC 23 399CGCUGCCUGCUGUGCCCCC 23 417 GGGGGCACAGCAGGCAGCG 120  417CCCAGCAGUGGGCCUUGUC 24 417 CCCAGCAGUGGGCCUUGUC 24 435GACAAGGCCCACUGCUGGG 121  435 CUGCUGGGAACGGCUCGUC 25 435CUGCUGGGAACGGCUCGUC 25 453 GACGAGCCGUUCCCAGCAG 122  453CAGAGGUGGAAGUGGUACC 26 453 CAGAGGUGGAAGUGGUACC 26 471GGUACCACUUCCACCUCUG 123  471 CCUUCCAGGAAGUGUGGGG 27 471CCUUCCAGGAAGUGUGGGG 27 489 CCCCACACUUCCUGGAAGG 124  489GCCGCAGCUACUGCCGGGC 28 489 GCCGCAGCUACUGCCGGGC 28 507GCCCGGCAGUAGCUGCGGC 125  507 CGCUGGAGAGGCUGGUGGA 29 507CGCUGGAGAGGCUGGUGGA 29 525 UCCACCAGCCUCUCCAGCG 126  525ACGUCGUGUCCGAGUACCC 30 525 ACGUCGUGUCCGAGUACCC 30 543GGGUACUCGGACACGACGU 127  543 CCAGCGAGGUGGAGCACAU 31 543CCAGCGAGGUGGAGCACAU 31 561 AUGUGCUCCACCUCGCUGG 128  561UGUUCAGCCCAUCCUGUGU 32 561 UGUUCAGCCCAUCCUGUGU 32 579ACACAGGAUGGGCUGAACA 129  579 UCUCCCUGCUGCGCUGCAC 33 579UCUCCCUGCUGCGCUGCAC 33 597 GUGCAGCGCAGCAGGGAGA 130  597CCGGCUGCUGCGGCGAUGA 34 597 CCGGCUGCUGCGGCGAUGA 34 615UCAUCGCCGCAGCAGCCGG 131  615 AGAAUCUGCACUGUGUGCC 35 615AGAAUCUGCACUGUGUGCC 35 633 GGCACACAGUGCAGAUUCU 132  633CGGUGGAGACGGCCAAUGU 36 633 CGGUGGAGACGGCCAAUGU 36 651ACAUUGGCCGUCUCCACCG 133  651 UCACCAUGCAGCUCCUAAA 37 651UCACCAUGCAGCUCCUAAA 37 669 UUUAGGAGCUGCAUGGUGA 134  669AGAUCCGUUCUGGGGACCG 38 669 AGAUCCGUUCUGGGGACCG 38 687CGGUCCCCAGAACGGAUCU 135  687 GGCCCUCCUACGUGGAGCU 39 687GGCCCUCCUACGUGGAGCU 39 705 AGCUCCACGUAGGAGGGCC 136  705UGACGUUCUCUCAGCACGU 40 705 UGACGUUCUCUCAGCACGU 40 723ACGUGCUGAGAGAACGUCA 137  723 UUCGCUGCGAAUGCCGGCC 41 723UUCGCUGCGAAUGCCGGCC 41 741 GGCCGGCAUUCGCAGCGAA 138  741CUCUGCGGGAGAAGAUGAA 42 741 CUCUGCGGGAGAAGAUGAA 42 759UUCAUCUUCUCCCGCAGAG 139  759 AGCCGGAAAGGAGGAGACC 43 759AGCCGGAAAGGAGGAGACC 43 777 GGUCUCCUCCUUUCCGGCU 140  777CCAAGGGCAGGGGGAAGAG 44 777 CCAAGGGCAGGGGGAAGAG 44 795CUCUUCCCCCUGCCCUUGG 141  795 GGAGGAGAGAGAAGCAGAG 45 795GGAGGAGAGAGAAGCAGAG 45 813 CUCUGCUUCUCUCUCCUCC 142  813GACCCACAGACUGCCACCU 46 813 GACCCACAGACUGCCACCU 46 831AGGUGGCAGUCUGUGGGUC 143  831 UGUGCGGCGAUGCUGUUCC 47 831UGUGCGGCGAUGCUGUUCC 47 849 GGAACAGCAUCGCCGCACA 144  849CCCGGAGGUAACCCACCCC 48 849 CCCGGAGGUAACCCACCCC 48 867GGGGUGGGUUACCUCCGGG 145  867 CUUGGAGGAGAGAGACCCC 49 867CUUGGAGGAGAGAGACCCC 49 885 GGGGUCUCUCUCCUCCAAG 146  885CGCACCCGGCUCGUGUAUU 50 885 CGCACCCGGCUCGUGUAUU 50 903AAUACACGAGCCGGGUGCG 147  903 UUAUUACCGUCACACUCUU 51 903UUAUUACCGUCACACUCUU 51 921 AAGAGUGUGACGGUAAUAA 148  921UCAGUGACUCCUGCUGGUA 52 921 UCAGUGACUCCUGCUGGUA 52 939UACCAGCAGGAGUCACUGA 149  939 ACCUGCCCUCUAUUUAUUA 53 939ACCUGCCCUCUAUUUAUUA 53 957 UAAUAAAUAGAGGGCAGGU 150  957AGCCAACUGUUUCCCUGCU 54 957 AGCCAACUGUUUCCCUGCU 54 975AGCAGGGAAACAGUUGGCU 151  975 UGAAUGCCUCGCUCCCUUC 55 975UGAAUGCCUCGCUCCCUUC 55 993 GAAGGGAGCGAGGCAUUCA 152  993CAAGACGAGGGGCAGGGAA 56 993 CAAGACGAGGGGCAGGGAA 56 1011UUCCGUGCCCCUCGUCUUG 153 1011 AGGACAGGACCCUCAGGAA 57 1011AGGACAGGACCCUCAGGAA 57 1029 UUCCUGAGGGUCCUGUCCU 154 1029AUUCAGUGCCUUCAACAAC 58 1029 AUUCAGUGCCUUCAACAAC 58 1047GUUGUUGAAGGCACUGAAU 155 1047 CGUGAGAGAAAGAGAGAAG 59 1047CGUGAGAGAAAGAGAGAAG 59 1065 CUUCUCUGUUUCUCUCACG 156 1065GCCAGCCACAGACCCCUGG 60 1065 GCCAGCCACAGACCCCUGG 60 1083CCAGGGGUCUGUGGCUGGC 157 1083 GGAGCUUCCGCUUUGAAAG 61 1083GGAGCUUCCGCUUUGAAAG 61 1101 CUUUCAAAGCGGAAGCUCC 158 1101GAAGCAAGACACGUGGCCU 62 1101 GAAGCAAGACACGUGGCCU 62 1119AGGCCACGUGUCUUGCUUC 159 1119 UCGUGAGGGGCAAGCUAGG 63 1119UCGUGAGGGGCAAGCUAGG 63 1137 CCUAGCUUGCCCCUCACGA 160 1137GCCCCAGAGGCCCUGGAGG 64 1137 GCCCCAGAGGCCCUGGAGG 64 1155CCUCCAGGGCCUCUGGGGC 161 1155 GUCUCCAGGGGCCUGCAGA 65 1155GUCUCCAGGGGCCUGCAGA 65 1173 UCUGCAGGCCCCUGGAGAC 162 1173AAGGAAAGAAGGGGGCCCU 66 1173 AAGGAAAGAAGGGGGCCCU 66 1191AGGGCCCCCUUCUUUCCUU 163 1191 UGCUACCUGUUCUUGGGCC 67 1191UGCUACCUGUUCUUGGGCC 67 1209 GGCCCAAGAACAGGUAGCA 164 1209CUCAGGCUCUGCACAGUCA 68 1209 CUCAGGCUCUGCACAGUCA 68 1227UGACUGUGCAGAGCCUGAG 165 1227 AAGCAGCCCUUGCUUUCGG 69 1227AAGCAGCCCUUGCUUUCGG 69 1245 CCGAAAGCAAGGGCUGCUU 166 1245GAGCUCCUGUCCAAAAGUA 70 1245 GAGCUCCUGUCCAAAAGUA 70 1263UACUUUUGGACAGGAGCUC 167 1263 AGGGAUGCGGAUCCUGCUG 71 1263AGGGAUGCGGAUCCUGCUG 71 1281 CAGCAGGAUCCGCAUCCCU 168 1281GGGGCCGCCACGGCCUGGC 72 1281 GGGGCCGCCACGGCCUGGC 72 1299GCCAGGCCGUGGCGGCCCC 169 1299 CUGGUGGGAAGGCCGGCAG 73 1299CUGGUGGGAAGGCCGGCAG 73 1317 CUGCCGGCCUUCCCACCAG 170 1317GCGGGCGGAGGGGAUCCAG 74 1317 GCGGGCGGAGGGGAUCCAG 74 1335CUGGAUCCCCUCCGCCCGC 171 1335 GCCACUUCCCCCUCUUCUU 75 1335GCCACUUCCCCCUCUUCUU 75 1353 AAGAAGAGGGGGAAGUGGC 172 1353UCUGAAGAUCAGAACAUUC 76 1353 UCUGAAGAUCAGAACAUUC 76 1371GAAUGUUCUGAUCUUCAGA 173 1371 CAGCUCUGGAGAACAGUGG 77 1371CAGCUCUGGAGAACAGUGG 77 1389 CCACUGUUCUCCAGAGCUG 174 1389GUUGCCUGGGGGCUUUUGC 78 1389 GUUGCCUGGGGGCUUUUGC 78 1407GCAAAAGCCCCCAGGCAAC 175 1407 CCACUCCUUGUCCCCCGUG 79 1407CCACUCCUUGUCCCCCGUG 79 1425 CACGGGGGACAAGGAGUGG 176 1425GAUCUCCCCUCACACUUUG 80 1425 GAUCUCCCCUCACACUUUG 80 1443CAAAGUGUGAGGGGAGAUC 177 1443 GCCAUUUGCUUGUACUGGG 81 1443GCCAUUUGCUUGUACUGGG 81 1461 CCCAGUACAAGCAAAUGGC 178 1461GACAUUGUUCUUUCCGGCC 82 1461 GACAUUGUUCUUUCCGGCC 82 1479GGCCGGAAAGAACAAUGUC 179 1479 CAAGGUGCCACCACCCUGC 83 1479CAAGGUGCCACCACCCUGC 83 1497 GCAGGGUGGUGGCACCUUG 180 1497CCCCCCCUAAGAGACACAU 84 1497 CCCCCCCUAAGAGACACAU 84 1515AUGUGUCUCUUAGGGGGGG 181 1515 UACAGAGUGGGCCCCGGGC 85 1515UACAGAGUGGGCCCCGGGC 85 1533 GCCCGGGGCCCACUCUGUA 182 1533CUGGAGAAAGAGCUGCCUG 86 1533 CUGGAGAAAGAGCUGCCUG 86 1551CAGGCAGCUCUUUCUCCAG 183 1551 GGAUGAGAAACAGCUCAGC 87 1551GGAUGAGAAACAGCUCAGC 87 1569 GCUGAGCUGUUUCUCAUCC 184 1569CCAGUGGGGAUGAGGUCAC 88 1569 CCAGUGGGGAUGAGGUCAC 88 1587GUGACCUCAUCCCCACUGG 185 1587 CCAGGGGAGGAGCCUGUGC 89 1587CCAGGGGAGGAGCCUGUGC 89 1605 GCACAGGCUCCUCCCCUGG 186 1605CGUCCCAGCUGAAGGCAGU 90 1605 CGUCCCAGCUGAAGGCAGU 90 1623ACUGCCUUCAGCUGGGACG 187 1623 UGGCAGGGGAGCAGGUUCC 91 1623UGGCAGGGGAGCAGGUUCC 91 1641 GGAACCUGCUCCCCUGCCA 188 1641CCCAAGGGCCCUGGCACCC 92 1641 CCCAAGGGCCCUGGCACCC 92 1659GGGUGCCAGGGCCCUUGGG 189 1659 CCCACAAGCUGUCCCUGCA 93 1659CCCACAAGCUGUCCCUGCA 93 1677 UGCAGGGACAGCUUGUGGG 190 1677AGGGCCAUCUGACUGCCAA 94 1677 AGGGCCAUCUGACUGCCAA 94 1695UUGGCAGUCAGAUGGCCCU 191 1695 AGCCAGAUUCUCUUGAAUA 95 1695AGCCAGAUUCUCUUGAAUA 95 1713 UAUUCAAGAGAAUCUGGCU 192 1713AAAGUAUUCUAGUGUGGAA 96 1713 AAAGUAUUCUAGUGUGGAA 96 1731UUCCACACUAGAAUACUUU 193 1724 GUGUGGAAAAAAAAAAAAA 97 1724GUGUGGAAAAAAAAAAAAA 97 1742 UUUUUUUUUUUUUCCACAC 194 The 3′-ends of theUpper sequence and the Lower sequence of the siNA construct can includean overhang sequence, for example about 1, 2, 3, or 4 nucleotides inlength, preferably 2 nucleotides in length, wherein the overhangingsequence of the lower sequence is optionally complementary to a portionof the target sequence. The upper sequence is also referred to as thesense strand, whereas the lower sequence is also referred to as theantisense strand. The upper and lower sequences in the Table can furthercomprise a chemical modification having Formulae I-VII, such asexemplary siNA constructs shown in FIGS. 4 and 5, or havingmodifications described in Table IV or any combination thereof.

TABLE III PGF Synthetic Modified siNA Constructs Target Seq Cmpd Seq PosTarget ID # Aliases Sequence ID  254 CACUCAGCUCUUCUCCUCCUGUG 195 PGF:256U21 sense siNA CUCAGCUCUUCUCCUCCUGTT 203  448 CUCGUCAGAGGUGGAAGUGGUAC196 PGF: 450U21 sense siNA CGUCAGAGGUGGAAGUGGUTT 204  648AUGUCACCAUGCAGCUCCUAAAG 197 PGF: 650U21 sense siNA GUCACCAUGCAGCUCCUAATT205  653 ACCAUGCAGCUCCUAAAGAUCCG 198 PGF: 655U21 sense siNACAUGCAGCUCCUAAAGAUCTT 206 1084 GAGCUUCCGCUUUGAAAGAAGCA 199 PGF: 1086U21sense siNA GCUUCCGCUUUGAAAGAAGTT 207 1091 CGCUUUGAAAGAAGCAAGACACG 200PGF: 1093U21 sense siNA CUUUGAAAGAAGCAAGACATT 208 1235CUUGCUUUCGGAGCUCCUGUCCA 201 PGF: 1237U21 sense siNAUGCUUUCGGAGCUCCUGUCTT 209 1539 AAAGAGCUGCCUGGAUGAGAAAC 202 PGF: 1541U21sense siNA AGAGCUGCCUGGAUGAGAATT 210  254 CACUCAGCUCUUCUCCUCCUGUG 195PGF: 274L21 antisense siNA CAGGAGGAGAAGAGCUGAGTT 211 (256C)  448CUCGUCAGAGGUGGAAGUGGUAC 196 PGF: 468L21 antisense siNAACCACUUCCACCUCUGACGTT 212 (450C)  648 AUGUCACCAUGCAGCUCCUAAAG 197 PGF:668L21 antisense siNA UUAGGAGCUGCAUGGUGACTT 213 (650C)  653ACCAUGCAGCUCCUAAAGAUCCG 198 PGF: 673L21 antisense siNAGAUCUUUAGGAGCUGCAUGTT 214 (655C) 1084 GAGCUUCCGCUUUGAAAGAAGCA 199 PGF:1104L21 antisense siNA CUUCUUUCAAAGCGGAAGCTT 215 (1086C) 1091CGCUUUGAAAGAAGCAAGACACG 200 PGF: 1111L21 antisense siNAUGUCUUGCUUCUUUCAAAGTT 216 (1093C) 1235 CUUGCUUUCGGAGCUCCUGUCCA 201 PGF:1255L21 antisense siNA GACAGGAGCUCCGAAAGCATT 217 (1237C) 1539AAAGAGCUGCCUGGAUGAGAAAC 202 PGF: 1559L21 antisense siNAUUCUCAUCCAGGCAGCUCUTT 218 (1541C)  254 CACUCAGCUCUUCUCCUCCUGUG 195 PGF:256U21 sense siNA stab04 B cucAGcucuucuccuccuGTT B 219  448CUCGUCAGAGGUGGAAGUGGUAC 196 PGF: 450U21 sense siNA stab04 BcGucAGAGGuGGAAGuGGuTT B 220  648 AUGUCACCAUGGAGCUCCUAAAG 197 PGF: 650U21sense siNA stab04 B GucAccAuGcAGcuccuAATT B 221  653ACCAUGCAGCUCCUAAAGAUCCG 198 PGF: 655U21 sense siNA stab04 BcAuGcAGcuccuAAAGAucTT B 222 1084 GAGCUUCCGCUUUGAAAGAAGCA 199 PGF:1086U21 sense siNA stab04 B GcuuccGcuuuGAAAGAAGTT B 223 1091CGCUUUGAAAGAAGCAAGACACG 200 PGF: 1093U21 sense siNA stab04 BcuuuGAAAGAAGcAAGAcATT B 224 1235 CUUGCUUUCGGAGCUCCUGUCCA 201 PGF:1237U21 sense siNA stab04 B uGcuuucGGAGcuccuGucTT B 225 1539AAAGAGCUGCCUGGAUGAGAAAC 202 PGF: 1541U21 sense siNA stab04 BAGAGcuGccuGGAuGAGAATT B 226  254 CACUCAGCUCUUCUCCUCCUGUG 195 PGF: 274L21antisense siNA cAGGAGGAGAAGAGcuGAGTsT 227 (256C) stab05  448CUCGUCAGAGGUGGAAGUGGUAC 196 PGF: 468L21 antisense siNAAccAcuuccAccucuGAcGTsT 228 (450C) stab05  648 AUGUCACCAUGCAGCUCCUAAAG197 PGF: 668L21 antisense siNA uuAGGAGcuGcAuGGuGAcTsT 229 (650C) stab05 653 ACCAUGCAGCUCCUAAAGAUCCG 198 PGF: 673L21 antisense siNAGAucuuuAGGAGcuGcAuGTsT 230 (655C) stab05 1084 GAGCUUCCGCUUUGAAAGAAGCA199 PGF: 1104L21 antisense siNA cuucuuucAAAGcGGAAGcTsT 231 (1086C)stab05 1091 CGCUUUGAAAGAAGCAAGACACG 200 PGF: 1111L21 antisense siNAuGucuuGcuucuuucAAAGTsT 232 (1093C) stab05 1235 CUUGCUUUCGGAGCUCCUGUCCA201 PGF: 1255L21 antisense siNA GAcAGGAGcuccGAAAGcATsT 233 (1237C)stab05 1539 AAAGAGCUGCCUGGAUGAGAAAC 202 PGF: 1559L21 antisense siNAuucucAuccAGGcAGcucuTsT 234 (1541C) stab05  254 CACUCAGCUCUUCUCCUCCUGUG195 PGF: 256U21 sense siNA stab07 B cucAGcucuucuccuccuGTT B 235  448CUCGUCAGAGGUGGAAGUGGUAC 196 PGF: 450U21 sense siNA stab07 BcGucAGAGGuGGAAGuGGuTT B 236  648 AUGUCACCAUGCAGCUCCUAAAG 197 PGF: 650U21sense siNA stab07 B GucAccAuGcAGcuccuAATT B 237  653ACCAUGCAGCUCCUAAAGAUCCG 198 PGF: 655U21 sense siNA stab07 BcAuGcAGcuccuAAAGAucTT B 238 1084 GAGCUUCCGCUUUGAAAGAAGCA 199 PGF:1086U21 sense siNA stab07 B GcuuccGcuuuGAAAGAAGTT B 239 1091CGCUUUGAAAGAAGCAAGACACG 200 PGF: 1093U21 sense siNA stab07 BcuuuGAAAGAAGcAAGAcATT B 240 1235 CUUGCUUUCGGAGCUCCUGUCCA 201 PGF:1237U21 sense siNA stab07 B uGcuuucGGAGcuccuGucTT B 241 1539AAAGAGCUGCCUGGAUGAGAAAC 202 PGF: 1541U21 sense siNA stab07 BAGAGcuGccuGGAuGAGAATT B 242  254 CACUCAGCUCUUCUCCUCCUGUG 195 PGF: 274L21antisense siNA cAGGAGGAGAAGAGcuGAGTsT 243 (256C) stab11  448CUCGUCAGAGGUGGAAGUGGUAC 196 PGF: 468L21 antisense siNAAccAcuuccAccucuGAcGTsT 244 (450C) stab11  648 AUGUCACCAUGCAGCUCCUAAAG197 PGF: 668L21 antisense siNA uuAGGAGcuGcAuGGuGAcTsT 245 (650C) stab11 653 ACCAUGCAGCUCCUAAAGAUCCG 198 PGF: 673L21 antisense siNAGAucuuuAGGAGcuGcAuGTsT 246 (655C) stab11 1084 GAGCUUCCGCUUUGAAAGAAGCA199 PGF: 1104L21 antisense siNA cuucuuucAAAGcGGAAGcTsT 247 (1086C)stab11 1091 CGCUUUGAAAGAAGCAAGACACG 200 PGF: 1111L21 antisense siNAuGucuuGcuucuuucAAAGTsT 248 (1093C) stab11 1235 CUUGCUUUCGGAGCUCCUGUCCA201 PGF: 1255L21 antisense siNA GAcAGGAGcuccGAAAGcATsT 249 (1237C)stab11 1539 AAAGAGCUGCCUGGAUGAGAAAC 202 PGF: 1559L21 antisense siNAuucucAuccAGGcAGcucuTsT 250 (1541C) stab11  254 CACUCAGCUCUUCUCCUCCUGUG195 PGF: 256U21 sense siNA stab18 B cucAGcucuucuccuccuGTT B 251  448CUCGUCAGAGGUGGAAGUGGUAC 196 PGF: 450U21 sense siNA stab18 BcGucAGAGGuGGAAGuGGuTT B 252  648 AUGUCACCAUGCAGCUCCUAAAG 197 PGF: 650U21sense siNA stab18 B GucAccAuGcAGcuccuAATT B 253  653ACCAUGCAGCUCCUAAAGAUCCG 198 PGF: 655U21 sense siNA stab18 BcAuGcAGcuccuAAAGAucTT B 254 1084 GAGCUUCCGCUUUGAAAGAAGCA 199 PGF:1086U21 sense siNA stab18 B GcuuccGcuuuGAAAGAAGTT B 255 1091CGCUUUGAAAGAAGCAAGACACG 200 PGF: 1093U21 sense siNA stab18 BcuuuGAAAGAAGcAAGAcATT B 256 1235 CUUGCUUUCGGAGCUCCUGUCCA 201 PGF:1237U21 sense siNA stab18 B uGcuuucGGAGcuccuGucTT B 257 1539AAAGAGCUGCCUGGAUGAGAAAC 202 PGF: 1541U21 sense siNA stab18 BAGAGcuGccuGGAuGAGAATT B 258  254 CACUCAGCUCUUCUCCUCCUGUG 195 34279 PGF:274L21 antisense siNA cAGGAGGAGAAGAGcuGAGTsT 259 (256C) stab08  448CUCGUCAGAGGUGGAAGUGGUAC 196 34280 PGF: 468L21 antisense siNAAccAcuuccAccucuGAcGTsT 260 (450C) stab08  648 AUGUCACCAUGCAGCUCCUAAAG197 34281 PGF: 668L21 antisense siNA uuAGGAGcuGcAuGGuGAcTsT 261 (650C)stab08  653 ACCAUGCAGCUCCUAAAGAUCCG 198 34282 PGF: 673L21 antisense siNAGAucuuuAGGAGcuGcAuGTsT 262 (655C) stab08 1084 GAGCUUCCGCUUUGAAAGAAGCA199 34283 PGF: 1104L21 antisense siNA cuucuuucAAAGcGGAAGcTsT 263 (1086C)stab08 1091 CGCUUUGAAAGAAGCAAGACACG 200 34284 PGF: 1111L21 antisensesiNA uGucuuGcuucuuucAAAGTsT 264 (1093C) stab08 1235CUUGCUUUCGGAGCUCCUGUCCA 201 34285 PGF: 1255L21 antisense siNAGAcAGGAGcuccGAAAGcATsT 265 (1237C) stab08 1539 AAAGAGCUGCCUGGAUGAGAAAC202 34286 PGF: 1559L21 antisense siNA uucucAuccAGGcAGcucuTsT 266 (1541C)stab08  254 CACUCAGCUCUUCUCCUCCUGUG 195 34263 PGF: 256U21 sense siNAstab09 B CUCAGCUCUUCUCCUCCUGTT B 267  448 CUCGUCAGAGGUGGAAGUGGUAC 19634264 PGF: 450U21 sense siNA stab09 B CGUCAGAGGUGGAAGUGGUTT B 268  648AUGUCACCAUGCAGCUCCUAAAG 197 34265 PGF: 650U21 sense siNA stab09 BGUCACCAUGCAGCUCCUAATT B 269  653 ACCAUGCAGCUCCUAAAGAUCCG 198 34266 PGF:655U21 sense siNA stab09 B CAUGCAGCUCCUAAAGAUCTT B 270 1084GAGCUUCCGCUUUGAAAGAAGCA 199 34267 PGF: 1086U21 sense siNA stab09 BGCUUCCGCUUUGAAAGAAGTT B 271 1091 CGCUUUGAAAGAAGCAAGACACG 200 34268 PGF:1093U21 sense siNA stab09 B CUUUGAAAGAAGCAAGACATT B 272 1235CUUGCUUUCGGAGCUCCUGUCCA 201 34269 PGF: 1237U21 sense siNA stab09 BUGCUUUCGGAGCUCCUGUCTT B 273 1539 AAAGAGCUGCCUGGAUGAGAAAC 202 34270 PGF:1541U21 sense siNA stab09 B AGAGCUGCCUGGAUGAGAATT B 274  254CACUCAGCUCUUCUCCUCCUGUG 195 34271 PGF: 274L21 antisense siNACAGGAGGAGAAGAGCUGAGTsT 275 (256C) stab10  448 CUCGUCAGAGGUGGAAGUGGUAC196 34272 PGF: 468L21 antisense siNA ACCACUUCCACCUCUGACGTsT 276 (450C)stab10  648 AUGUCACCAUGCAGCUCCUAAAG 197 34273 PGF: 668L21 antisense siNAUUAGGAGCUGCAUGGUGACTsT 277 (650C) stab10  653 ACCAUGCAGCUCCUAAAGAUCCG198 34274 PGF: 673L21 antisense siNA GAUCUUUAGGAGCUGCAUGTsT 278 (655C)stab10 1084 GAGCUUCCGCUUUGAAAGAAGCA 199 34275 PGF: 1104L21 antisensesiNA CUUCUUUCAAAGCGGAAGCTsT 279 (1086C) stab10 1091CGCUUUGAAAGAAGCAAGACACG 200 34276 PGF: 1111L21 antisense siNAUGUCUUGCUUCUUUCAAAGTsT 280 (1093C) stab10 1235 CUUGCUUUCGGAGCUCCUGUCCA201 34277 PGF: 1255L21 antisense siNA GACAGGAGCUCCGAAAGCATsT 281 (1237C)stab10 1539 AAAGAGCUGCCUGGAUGAGAAAC 202 34278 PGF: 1559L21 antisensesiNA UUCUCAUCCAGGCAGCUCUTsT 282 (1541C) stab10  254CACUCAGCUCUUCUCCUCCUGUG 195 PGF: 274L21 antisense siNAcAGGAGGAGAAGAGcuGAGTT B 283 (256C) stab19  448 CUCGUCAGAGGUGGAAGUGGUAC196 PGF: 468L21 antisense siNA AccAcuuccAccucuGAcGTT B 284 (450C) stab19 648 AUGUCACCAUGCAGCUCCUAAAG 197 PGF: 668L21 antisense siNAuuAGGAGcuGcAuGGuGAcTT B 285 (650C) stab19  653 ACCAUGCAGCUCCUAAAGAUCCG198 PGF: 673L21 antisense siNA GAucuuuAGGAGcuGcAuGTT B 286 (655C) stab191084 GAGCUUCCGCUUUGAAAGAAGCA 199 PGF: 1104L21 antisense siNAcuucuuucAAAGcGGAAGcTT B 287 (1086C) stab19 1091 CGCUUUGAAAGAAGCAAGACACG200 PGF: 1111L21 antisense siNA uGucuuGcuucuuucAAAGTT B 288 (1093C)stab19 1235 CUUGCUUUCGGAGCUCCUGUCCA 201 PGF: 1255L21 antisense siNAGAcAGGAGcuccGAAAGcATT B 289 (1237C) stab19 1539 AAAGAGCUGCCUGGAUGAGAAAC202 PGF: 1559L21 antisense siNA uucucAuccAGGcAGcucuTT B 290 (1541C)stab19  254 CACUCAGCUCUUCUCCUCCUGUG 195 PGF: 274L21 antisense siNACAGGAGGAGAAGAGCUGAGTT B 291 (256C) stab22  448 CUCGUCAGAGGUGGAAGUGGUAC196 PGF: 468L21 antisense siNA ACCACUUCCACCUCUGACGTT B 292 (450C) stab22 648 AUGUCACCAUGCAGCUCCUAAAG 197 PGF: 668L21 antisense siNAUUAGGAGCUGCAUGGUGACTT B 293 (650C) stab22  653 ACCAUGCAGCUCCUAAAGAUCCG198 PGF: 673L21 antisense siNA GAUCUUUAGGAGCUGCAUGTT B 294 (655C) stab221084 GAGCUUCCGCUUUGAAAGAAGCA 199 PGF: 1104L21 antisense siNACUUCUUUCAAAGCGGAAGCTT B 295 (1086C) stab22 1091 CGCUUUGAAAGAAGCAAGACACG200 PGF: 1111L21 antisense siNA UGUCUUGCUUCUUUCAAAGTT B 296 (1093C)stab22 1235 CUUGCUUUCGGAGCUCCUGUCCA 201 PGF: 1255L21 antisense siNAGACAGGAGCUCCGAAAGCATT B 297 (1237C) stab22 1539 AAAGAGCUGCCUGGAUGAGAAAC202 PGF: 1559L21 antisense siNA UUCUCAUCCAGGCAGCUCUTT B 298 (1541C)stab22 Uppercase = ribonucleotide u, c = 2′-deoxy-2′-fluoro U, C T= thymidine B = inverted deoxy abasic s = phosphorothioate linkageA = deoxy Adenosine G = deoxy Guanosine G = 2′-O-methyl GuanosineA = 2′-O-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chem- Pu- istry pyrimidine rine capp = S Strand “Stab Ribo Ribo TT at 3′- S/AS 00” ends “Stab Ribo Ribo — 5at 5′-end S/AS 1” 1 at 3′-end “Stab Ribo Ribo — All linkages Usually AS2” “Stab 2′-fluoro Ribo — 4 at 5′-end Usually S 3” 4 at 3′-end “Stab2′-fluoro Ribo 5′ and 3′- — Usually S 4” ends “Stab 2′-fluoro Ribo — 1at 3′-end Usually AS 5” “Stab 2′-O-Methyl Ribo 5′ and 3′- — Usually S 6”ends “Stab 2′-fluoro 2′- 5′ and 3′- — Usually S 7” deoxy ends “Stab2′-fluoro 2′-O- — 1 at 3′-end S/AS 8” Methyl “Stab Ribo Ribo 5′ and 3′-— Usually S 9” ends “Stab Ribo Ribo — 1 at 3′-end Usually AS 10” “Stab2′-fluoro 2′- — 1 at 3′-end Usually AS 11” deoxy “Stab 2′-fluoro LNA 5′and 3′- Usually S 12” ends “Stab 2′-fluoro LNA 1 at 3′-end Usually AS13” “Stab 2′-fluoro 2′- 2 at 5′-end Usually AS 14” deoxy 1 at 3′-end“Stab 2′-deoxy 2′- 2 at 5′-end Usually AS 15” deoxy 1 at 3′-end “StabRibo 2′-O- 5′ and 3′- Usually S 16” Methyl ends “Stab 2′-O-Methyl 2′-O-5′ and 3′- Usually S 17” Methyl ends “Stab 2′-fluoro 2′-O- 5′ and 3′-Usually S 18” Methyl ends “Stab 2′-fluoro 2′-O- 3′-end S/AS 19” Methyl“Stab 2′-fluoro 2′- 3′-end Usually AS 20” deoxy “Stab 2′-fluoro Ribo3′-end Usually AS 21” “Stab Ribo Ribo 3′-end Usually AS 22” “Stab2′-fluoro* 2′- 5′ and 3′- Usually S 23” deoxy* ends “Stab 2′-fluoro*2′-O- — 1 at 3′-end S/AS 24” Methyl* “Stab 2′-fluoro* 2′-O- — 1 at3′-end S/AS 25” Methyl* “Stab 2′-fluoro* 2′-O- — S/AS 26” Methyl* “Stab2′-fluoro* 2′-O- 3′-end S/AS 27” Methyl* “Stab 2′-fluoro* 2′-O- 3′-endS/AS 28” Methyl* “Stab 2′-fluoro* 2′-O- 1 at 3′-end S/AS 29” Methyl*“Stab 2′-fluoro* 2′-O- S/AS 30” Methyl* “Stab 2′-fluoro* 2′-O- 3′-endS/AS 31” Methyl* “Stab 2′-fluoro 2′-O- S/AS 32” Methyl CAP = anyterminal cap, see for example FIG. 10. All Stab 00-32 chemistries cancomprise 3′-terminal thymidine (TT) residues All Stab 00-32 chemistriestypically comprise about 21 nucleotides, but can vary as describedherein. S = sense strand AS = antisense strand *Stab 23 has a singleribonucleotide adjacent to 3′-CAP *Stab 24 and Stab 28 have a singleribonucleotide at 5′-terminus *Stab 25, Stab 26, and Stab 27 have threeribonucleotides at 5′-terminus *Stab 29, Stab 30, and Stab 31, anypurine at first three nucleotide positions from 5′-terminus areribonucleotides p = phosphorothioate linkage

TABLE V A. 2.5 μmol Synthesis Cycle ABI 394 Instrument ReagentEquivalents Amount Wait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNAPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Reagent Equivalents AmountWait Time* DNA Wait Time* 2′-O-methyl Wait Time*RNA Phosphoramidites 1531 μL 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min465 sec Acetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124μL 5 sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 secIodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle96 well Instrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* 2′-O-Reagent 2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time*Ribo Phosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 secS-Ethyl Tetrazole  70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified short interfering nucleic acid (siNA) molecule, wherein: (a) the siNA molecule comprises a sense strand and a separate antisense strand, each strand having one or more pyrimidine nucleotides and one or more purine nucleotides; (b) each strand is independently 18 to 23 nucleotides in length, and the sense strand is complementary to the antisense strand; (c) the antisense strand is complementary to a human placental growth factor 1 (PGF-1) RNA sequence comprising SEQ ID NO: 321; (d) a plurality of the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and a plurality of the purine nucleotides present in the sense strand are 2′-deoxy purine nucleotides; and, (e) a plurality of the pyrimidine nucleotides in the antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and a plurality of the purine nucleotides present in the antisense strand are 2′-O-methyl purine nucleotides.
 2. The siNA molecule of claim 1, wherein the sense strand includes a terminal cap moiety at both 5′- and 3′- ends.
 3. The siNA molecule of claim 1, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise a 3′-overhang.
 4. A composition comprising the siNA molecule of claim 1 and a pharmaceutically acceptable carrier or diluent.
 5. The siNA molecule of claim 1, wherein the antisense strand has a phosphorothioate internucleotide linkage at the 3′- end. 